Thymic stromal lymphopoietin receptor molecules and uses thereof

ABSTRACT

The present invention provides Thymic Stromal Lymphopoietin Receptor (TSLPR) polypeptides and nucleic acid molecules encoding the same. The invention also provides selective binding agents, vectors, host cells, and methods for producing TSLPR polypeptides. The invention further provides pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, disorders, and conditions associated with TSLPR polypeptides.

[0001] This application claims the benefit of priority from U.S.Provisional Patent Application No. 60/214,866, filed on Jun. 28, 2000,the disclosure of which is explicitly incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to Thymic Stromal LymphopoietinReceptor (TSLPR) polypeptides and nucleic acid molecules encoding thesame. The invention also relates to selective binding agents, vectors,host cells, and methods for producing TSLPR polypeptides. The inventionfurther relates to pharmaceutical compositions and methods for thediagnosis, treatment, amelioration, and/or prevention of diseases,disorders, and conditions associated with TSLPR polypeptides.

BACKGROUND OF THE INVENTION

[0003] Technical advances in the identification, cloning, expression,and manipulation of nucleic acid molecules and the deciphering of thehuman genome have greatly accelerated the discovery of noveltherapeutics. Rapid nucleic acid sequencing techniques can now generatesequence information at unprecedented rates and, coupled withcomputational analyses, allow the assembly of overlapping sequences intopartial and entire genomes and the identification ofpolypeptide-encoding regions. A comparison of a predicted amino acidsequence against a database compilation of known amino acid sequencesallows one to determine the extent of homology to previously identifiedsequences and/or structural landmarks. The cloning and expression of apolypeptide-encoding region of a nucleic acid molecule provides apolypeptide product for structural and functional analyses. Themanipulation of nucleic acid molecules and encoded polypeptides mayconfer advantageous properties on a product for use as a therapeutic

[0004] In spite of the significant technical advances in genome researchover the past decade, the potential for the development of noveltherapeutics based on the human genome is still largely unrealized. Manygenes encoding potentially beneficial polypeptide therapeutics or thoseencoding polypeptides, which may act as “targets” for therapeuticmolecules, have still not been identified. Accordingly, it is an objectof the invention to identify novel polypeptides, and nucleic acidmolecules encoding the same, which have diagnostic or therapeuticbenefit.

[0005] Cytokines regulate a variety of cellular responses includingproliferation, differentiation, and survival. Among the differentclasses of cytokines are the type I cytokines, which form four α-helicalbundle structures that exhibit an up-up-down-down topology (Bazan, 1990,Immunol. Today 11:350-54; Leonard and O'Shea, 1998, Annu. Rev. Immunol.16:293-322; Leonard, Fundamental Immunology 741-74 (Paul, ed.,Lippincott Raven Publishers 4 ed., 1999)). Type I cytokines include manyinterleukins, such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11,IL-12, IL-13, and IL-15 as well as other hematologically-activemolecules such as GM-CSF, erythropoietin, tbrombopoietin, and moleculessuch as growth hormone and prolactin. Signaling by type I cytokinesinvolves interaction with homodimers, heterodimers, or higher orderreceptor oligomers of the type I cytokine receptor superfamily. Ligandbinding induces dimerization or higher order oligomerization, resultingin downstream signaling, in part involving the Jak-STAT pathway (Bazan,supra; Leonard and O'Shea, supra; Leonard, supra).

[0006] Thymic stromal lymphopoietin (TSLP) is a cytokine whosebiological activities overlap with those of IL-7. For example, both TSLPand IL-7 induce tyrosine phosphorylation of the transcription factorStat5 (Isaksen et al., 1999, J. Immunol. 163:5971-77). TSLP activity wasoriginally identified in the conditioned medium of a thymic stromal cellline that supported the development of murine IgM⁺ B-cells from fetalliver hematopoietic progenitor cells (Friend et al., 1994 Exp. Hematol.22:321-28). Moreover, TSLP can promote B-cell lymphopoiesis in long termbone marrow cultures and can co-stimulate both thymocytes and matureT-cells (Friend et al., supra; Levin et al., 1999, J. Immunol.162:677-83). TSLP may also serve as an extrinsic signal to specificallyrearrange the T-cell receptor gamma locus (Candeias et al., 1997,Immunol. Lett. 57:9-14). Thus, the isolation and characterization of thecytokine receptor for TSLP would allow for the identification ofcompounds useful in treating TSLP-related diseases or conditions, suchas those affecting B-cell development, T-cell development, T-cellreceptor gene rearrangement, or regulation of the Stat5 transcriptionfactor.

SUMMARY OF THE INVENTION

[0007] The present invention relates to novel TSLPR nucleic acidmolecules and encoded polypeptides.

[0008] The invention provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

[0009] (a) the nucleotide sequence as set forth in any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11;

[0010] (b) a nucleotide sequence encoding the polypeptide as set forthin any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0011] (c) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of either (b) or (c); and

[0012] (d) a nucleotide sequence complementary to either (b) or (c).

[0013] The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

[0014] (a) a nucleotide sequence encoding a polypeptide which is atleast about 70 percent identical to the polypeptide as set forth in anyof SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, wherein the encodedpolypeptide has an activity of the polypeptide set forth in any of SEQID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0015] (b) a nucleotide sequence encoding an allelic variant or splicevariant of the nucleotide sequence as set forth in any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 1, or (a);

[0016] (c) a region of the nucleotide sequence of any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, (a), or (b)encoding a polypeptide fragment of at least about 25 amino acidresidues, wherein the polypeptide fragment has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8, or is antigenic;

[0017] (d) a region of the nucleotide sequence of any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, or any of(a)-(c) comprising a fragment of at least about 16 nucleotides;

[0018] (e) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of any of (a)-(d); and

[0019] (f) a nucleotide sequence complementary to any of (a)-(d).

[0020] The invention further provides for an isolated nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of:

[0021] (a) a nucleotide sequence encoding a polypeptide as set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least oneconservative amino acid substitution, wherein the encoded polypeptidehas an activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQID NO: 5, or SEQ ID NO: 8;

[0022] (b) a nucleotide sequence encoding a polypeptide as set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least oneamino acid insertion, wherein the encoded polypeptide has an activity ofthe polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQID NO: 8;

[0023] (c) a nucleotide sequence encoding a polypeptide as set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least oneamino acid deletion, wherein the encoded polypeptide has an activity ofthe polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQID NO: 8;

[0024] (d) a nucleotide sequence encoding a polypeptide as set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 which has a C- and/orN-terminal truncation, wherein the encoded polypeptide has an activityof the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, orSEQ ID NO: 8;

[0025] (e) a nucleotide sequence encoding a polypeptide as set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least onemodification selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the encoded polypeptidehas an activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQID NO: 5, or SEQ ID NO: 8;

[0026] (f) a nucleotide sequence of any of (a)-(e) comprising a fragmentof at least about 16 nucleotides;

[0027] (g) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of any of (a)-(f); and

[0028] (h) a nucleotide sequence complementary to any of (a)-(e).

[0029] The present invention provides for an isolated polypeptidecomprising the amino acid sequence as set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8.

[0030] The invention also provides for an isolated polypeptidecomprising the amino acid sequence selected from the group consistingof:

[0031] (a) the amino acid sequence as set forth in any of SEQ ID NO: 3,SEQ D NO: 6, or SEQ ID NO: 9, optionally further comprising anamino-terminal methionine;

[0032] (b) an amino acid sequence for an ortholog of any of SEQ ID NO:2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0033] (c) an amino acid sequence which is at least about 70 percentidentical to the amino acid sequence of any of SEQ ID NO: 2, SEQ ID NO:5, or SEQ ID NO: 8, wherein the polypeptide has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8;

[0034] (d) a fragment of the amino acid sequence set forth in any of SEQID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 comprising at least about 25amino acid residues, wherein the fragment has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8, or is antigenic; and

[0035] (e) an amino acid sequence for an allelic variant or splicevariant of the amino acid sequence as set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8, or any of (a)-(c).

[0036] The invention further provides for an isolated polypeptidecomprising the amino acid sequence selected from the group consistingof:

[0037] (a) the amino acid sequence as set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8 with at least one conservative amino acidsubstitution, wherein the polypeptide has an activity of the polypeptideset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0038] (b) the amino acid sequence as set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8 with at least one amino acid insertion,wherein the polypeptide has an activity of the polypeptide set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0039] (c) the amino acid sequence as set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8 with at least one amino acid deletion,wherein the polypeptide has an activity of the polypeptide set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0040] (d) the amino acid sequence as set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8 which has a C- and/or N-terminaltruncation, wherein the polypeptide has an activity of the polypeptideset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8; and

[0041] (e) the amino acid sequence as set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8 with at least one modification selectedfrom the group consisting of amino acid substitutions, amino acidinsertions, amino acid deletions, C-terminal truncation, and N-terminaltruncation, wherein the polypeptide has an activity of the polypeptideset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.

[0042] Also provided are fusion polypeptides comprising TSLPR amino acidsequences.

[0043] The present invention also provides for an expression vectorcomprising the isolated nucleic acid molecules as set forth herein,recombinant host cells comprising the recombinant nucleic acid moleculesas set forth herein, and a method of producing a TSLPR polypeptidecomprising culturing the host cells and optionally isolating thepolypeptide so produced.

[0044] A transgenic non-human animal comprising a nucleic acid moleculeencoding a TSLPR polypeptide is also encompassed by the invention. TheTSLPR nucleic acid molecules are introduced into the animal in a mannerthat allows expression and increased levels of a TSLPR polypeptide,which may include increased circulating levels. Alternatively, the TSLPRnucleic acid molecules are introduced into the animal in a manner thatprevents expression of endogenous TSLPR polypeptide (i.e., generates atransgenic animal possessing a TSLPR polypeptide gene knockout). Thetransgenic non-human animal is preferably a mammal, and more preferablya rodent, such as a rat or a mouse.

[0045] Also provided are derivatives of the TSLPR polypeptides of thepresent invention.

[0046] Additionally provided are selective binding agents such asantibodies and peptides capable of specifically binding the TSLPRpolypeptides of the invention. Such antibodies and peptides may beagonistic or antagonistic.

[0047] Pharmaceutical compositions comprising the nucleotides,polypeptides, or selective binding agents of the invention and one ormore pharmaceutically acceptable formulation agents are also encompassedby the invention. The pharmaceutical compositions are used to providetherapeutically effective amounts of the nucleotides or polypeptides ofthe present invention. The invention is also directed to methods ofusing the polypeptides, nucleic acid molecules, and selective bindingagents.

[0048] The TSLPR polypeptides and nucleic acid molecules of the presentinvention may be used to treat, prevent, ameliorate, and/or detectdiseases and disorders, including those recited herein.

[0049] The present invention also provides a method of assaying testmolecules to identify a test molecule that binds to a TSLPR polypeptide.The method comprises contacting a TSLPR polypeptide with a test moleculeto determine the extent of binding of the test molecule to thepolypeptide. The method further comprises determining whether such testmolecules are agonists or antagonists of a TSLPR polypeptide. Thepresent invention further provides a method of testing the impact ofmolecules on the expression of TSLPR polypeptide or on the activity ofTSLPR polypeptide.

[0050] Methods of regulating expression and modulating (i.e., increasingor decreasing) levels of a TSLPR polypeptide are also encompassed by theinvention. One method comprises administering to an animal a nucleicacid molecule encoding a TSLPR polypeptide. In another method, a nucleicacid molecule comprising elements that regulate or modulate theexpression of a TSLPR polypeptide may be administered. Examples of thesemethods include gene therapy, cell therapy, and anti-sense therapy asfurther described herein.

BRIEF DESCRIPTION OF THE FIGURES

[0051] FIGS. 1A-1B illustrate the nucleotide sequence of the murineTSLPR gene (SEQ ID NO: 1) and deduced amino acid sequence of murineTSLPR polypeptide (SEQ ID NO: 2). The predicted signal peptide(underline) and transmembrane domain (double underline) are indicated;

[0052]FIG. 2 illustrates an amino acid sequence alignment of murineTSLPR polypeptide (upper sequence; SEQ ID NO: 2) and murine commoncytokine receptor γ chain (γ_(c)) (lower sequence; SEQ ID NO: 12).Identical residues (boxed), potential N-linked glycosylation sites (*),and predicted signal peptide and transmembrane domain (underline) areindicated;

[0053] FIGS. 3A-3B illustrate the nucleotide sequence of the human TSLPRgene (SEQ ID NO: 4) and the deduced amino acid sequence of human TSLPRpolypeptide (SEQ ID NO: 5). The predicted signal peptide (underline) andtransmembrane domain (double underline) are indicated;

[0054] FIGS. 4A-4B illustrate the nucleotide sequence of humanTSLPR/FLAG (SEQ ID NO: 7) and the deduced amino acid sequence of humanTSLPR/FLAG polypeptide (SEQ ID NO: 8). The FLAG peptide (dottedunderline), predicted signal peptide (underline), and predictedtransmembrane domain (double underline) are indicated;

[0055]FIG. 5 illustrates an amino acid sequence alignment of murineTSLPR polypeptide (upper sequence; SEQ ID NO: 2) and human TSLPRpolypeptide (lower sequence; SEQ ID NO: 5);

[0056] FIGS. 6A-6C illustrate (A) in vitro translation of murine TSLPRpolypeptide, (B) immunoprecipitation of murine TSLPR polypeptide fromNAG 8/7 cells, and (C) northern blot analysis of murine TSLPR mRNAexpression.

[0057]FIG. 7 illustrates the results obtained in proliferation assaysusing cells transfected with chimeric expression constructs forc-Kit/γ_(c), c-Kit/TSLPR and c-Kit/β, or c-Kit/γ_(c) and c-Kit/β.

[0058] FIGS. 8A-8C illustrate the results obtained in affinity labelingassays in which ¹²⁵I-TSLP was added to 293 cells transfected withexpression constructs for murine IL-7Rα, murine TSLPR, murine IL-7Rα andmurine TSLPR, or human IL-7Rα and murine TSLPR, and then cross-linkedwith DSS.

[0059] FIGS. 9A-9D illustrate the results obtained in displacementbinding assays.

[0060]FIG. 10 illustrates the results obtained in CAT assays in whichHepG2 cells were co-transfected with expression constructs for IL-7Rαand TSLPR, or γ_(c), and pHRRE-CAT.

DETAILED DESCRIPTION OF THE INVENTION

[0061] The section headings used herein are for organizational purposesonly and are not to be construed as limiting the subject matterdescribed. All references cited in this application are expresslyincorporated by reference herein.

[0062] Definitions

[0063] The terms “TSLPR gene” or “TSLPR nucleic acid molecule” or “TSLPRpolynucleotide” refer to a nucleic acid molecule comprising orconsisting of a nucleotide sequence as set forth in any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, anucleotide sequence encoding the polypeptide as set forth in any of SEQID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, and nucleic acid molecules asdefined herein.

[0064] The term “TSLPR polypeptide allelic variant” refers to one ofseveral possible naturally occurring alternate forms of a gene occupyinga given locus on a chromosome of an organism or a population oforganisms.

[0065] The term “TSLPR polypeptide splice variant” refers to a nucleicacid molecule, usually RNA, which is generated by alternative processingof intron sequences in an RNA transcript of TSLPR polypeptide amino acidsequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8.

[0066] The term “isolated nucleic acid molecule” refers to a nucleicacid molecule of the invention that (1) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates, or other materialswith which it is naturally found when total nucleic acid is isolatedfrom the source cells, (2) is not linked to all or a portion of apolynucleotide to which the “isolated nucleic acid molecule” is linkedin nature, (3) is operably linked to a polynucleotide which it is notlinked to in nature, or (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

[0067] The term “nucleic acid sequence” or “nucleic acid molecule”refers to a DNA or RNA sequence. The term encompasses molecules formedfrom any of the known base analogs of DNA and RNA such as, but notlimited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0068] The term “vector” is used to refer to any molecule (e.g., nucleicacid, plasmid, or virus) used to transfer coding information to a hostcell.

[0069] The term “expression vector” refers to a vector that is suitablefor transformation of a host cell and contains nucleic acid sequencesthat direct and/or control the expression of inserted heterologousnucleic acid sequences. Expression includes, but is not limited to,processes such as transcription, translation, and RNA splicing, ifintrons are present.

[0070] The term “operably linked” is used herein to refer to anarrangement of flanking sequences wherein the flanking sequences sodescribed are configured or assembled so as to perform their usualfunction. Thus, a flanking sequence operably linked to a coding sequencemay be capable of effecting the replication, transcription and/ortranslation of the coding sequence. For example, a coding sequence isoperably linked to a promoter when the promoter is capable of directingtranscription of that coding sequence. A flanking sequence need not becontiguous with the coding sequence, so long as it functions correctlyThus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence can still be considered “operably linked” to thecoding sequence.

[0071] The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

[0072] The term “TSLPR polypeptide” refers to a polypeptide comprisingthe amino acid sequence of any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8 and related polypeptides. Related polypeptides include TSLPRpolypeptide fragments, TSLPR polypeptide orthologs, TSLPR polypeptidevariants, and TSLPR polypeptide derivatives, which possess at least oneactivity of the polypeptide as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, or SEQ ID NO: 8. TSLPR polypeptides may be mature polypeptides,as defined herein, and may or may not have an amino-terminal methionineresidue, depending on the method by which they are prepared.

[0073] The term “TSLPR polypeptide fragment” refers to a polypeptidethat comprises a truncation at the amino-terminus (with or without aleader sequence) and/or a truncation at the carboxyl-terminus of thepolypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8. The term “TSLPR polypeptide fragment” also refers toamino-terminal and/or carboxyl-terminal truncations of TSLPR polypeptideorthologs, TSLPR polypeptide derivatives, or TSLPR polypeptide variants,or to amino-terminal and/or carboxyl-terminal truncations of thepolypeptides encoded by TSLPR polypeptide allelic variants or TSLPRpolypeptide splice variants. TSLPR polypeptide fragments may result fromalternative RNA splicing or from in vivo protease activity.Membrane-bound forms of a TSLPR polypeptide are also contemplated by thepresent invention. In preferred embodiments, truncations and/ordeletions comprise about 10 amino acids, or about 20 amino acids, orabout 50 amino acids, or about 75 amino acids, or about 100 amino acids,or more than about 100 amino acids. The polypeptide fragments soproduced will comprise about 25 contiguous amino acids, or about 50amino acids, or about 75 amino acids, or about 100 amino acids, or about150 amino acids, or about 200 amino acids. Such TSLPR polypeptidefragments may optionally comprise an amino-terminal methionine residue.It will be appreciated that such fragments can be used, for example, togenerate antibodies to TSLPR polypeptides.

[0074] The term “TSLPR polypeptide ortholog” refers to a polypeptidefrom another species that corresponds to TSLPR polypeptide amino acidsequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8. For example, mouse and human TSLPR polypeptides are consideredorthologs of each other.

[0075] The term “TSLPR polypeptide variants” refers to TSLPRpolypeptides comprising amino acid sequences having one or more aminoacid sequence substitutions, deletions (such as internal deletionsand/or TSLPR polypeptide fragments), and/or additions (such as internaladditions and/or TSLPR fusion polypeptides) as compared to the TSLPRpolypeptide amino acid sequence set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, or SEQ ID NO: 8 (with or without a leader sequence). Variants maybe naturally occurring (e.g., TSLPR polypeptide allelic variants, TSLPRpolypeptide orthologs, and TSLPR polypeptide splice variants) orartificially constructed. Such TSLPR polypeptide variants may beprepared from the corresponding nucleic acid molecules having a DNAsequence that varies accordingly from the DNA sequence as set forth inany of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQID NO: 11. In preferred embodiments, the variants have from 1 to 3, orfrom 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or morethan 100 amino acid substitutions, insertions, additions and/ordeletions, wherein the substitutions may be conservative, ornon-conservative, or any combination thereof.

[0076] The term “TSLPR polypeptide derivatives” refers to thepolypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8, TSLPR polypeptide fragments, TSLPR polypeptide orthologs, orTSLPR polypeptide variants, as defined herein, that have been chemicallymodified. The term “TSLPR polypeptide derivatives” also refers to thepolypeptides encoded by TSLPR polypeptide allelic variants or TSLPRpolypeptide splice variants, as defined herein, that have beenchemically modified.

[0077] The term “mature TSLPR polypeptide” refers to a TSLPR polypeptidelacking a leader sequence. A mature TSLPR polypeptide may also includeother modifications such as proteolytic processing of the amino-terminus(with or without a leader sequence) and/or the carboxyl-terminus,cleavage of a smaller polypeptide from a larger precursor, N-linkedand/or O-linked glycosylation, and the like. Exemplary mature TSLPRpolypeptides are depicted by the amino acid sequences as set forth inSEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.

[0078] The term “TSLPR fusion polypeptide” refers to a fusion of one ormore amino acids (such as a heterologous protein or peptide) at theamino- or carboxyl-terminus of the polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, TSLPR polypeptidefragments, TSLPR polypeptide orthologs, TSLPR polypeptide variants, orTSLPR derivatives, as defined herein. The term “TSLPR fusionpolypeptide” also refers to a fusion of one or more amino acids at theamino- or carboxyl-terminus of the polypeptide encoded by TSLPRpolypeptide allelic variants or TSLPR polypeptide splice variants, asdefined herein.

[0079] The term “biologically active TSLPR polypeptides” refers to TSLPRpolypeptides having at least one activity characteristic of thepolypeptide comprising the amino acid sequence of any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8. In addition, a TSLPR polypeptide may beactive as an immunogen; that is, the TSLPR polypeptide contains at leastone epitope to which antibodies may be raised.

[0080] The term “isolated polypeptide” refers to a polypeptide of thepresent invention that (1) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is naturally found when isolated from the source cell, (2)is not linked (by covalent or noncovalent interaction) to all or aportion of a polypeptide to which the “isolated polypeptide” is linkedin nature, (3) is operably linked (by covalent or noncovalentinteraction) to a polypeptide with which it is not linked in nature, or(4) does not occur in nature. Preferably, the isolated polypeptide issubstantially free from any other contaminating polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its therapeutic, diagnostic, prophylactic or researchuse.

[0081] The term “identity,” as known in the art, refers to arelationship between the sequences of two or more polypeptide moleculesor two or more nucleic acid molecules, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness between nucleic acid molecules or polypeptides, as the casemay be, as determined by the match between strings of two or morenucleotide or two or more amino acid sequences. “Identity” measures thepercent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”).

[0082] The term “similarity” is a related concept, but in contrast to“identity,” “similarity” refers to a measure of relatedness whichincludes both identical matches and conservative substitution matches.If two polypeptide sequences have, for example, 10/20 identical aminoacids, and the remainder are all non-conservative substitutions, thenthe percent identity and similarity would both be 50%. If in the sameexample, there are five more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15/20). Therefore, in cases where there areconservative substitutions, the percent similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

[0083] The term “naturally occurring” or “native” when used inconnection with biological materials such as nucleic acid molecules,polypeptides, host cells, and the like, refers to materials which arefound in nature and are not manipulated by man. Similarly,“non-naturally occurring” or “non-native” as used herein refers to amaterial that is not found in nature or that has been structurallymodified or synthesized by man.

[0084] The terms “effective amount” and “therapeutically effectiveamount” each refer to the amount of a TSLPR polypeptide or TSLPR nucleicacid molecule used to support an observable level of one or morebiological activities of the TSLPR polypeptides as set forth herein.

[0085] The term “pharmaceutically acceptable carrier” or“physiologically acceptable carrier” as used herein refers to one ormore formulation materials suitable for accomplishing or enhancing thedelivery of the TSLPR polypeptide, TSLPR nucleic acid molecule, or TSLPRselective binding agent as a pharmaceutical composition.

[0086] The term “antigen” refers to a molecule or a portion of amolecule capable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

[0087] The term “selective binding agent” refers to a molecule ormolecules having specificity for a TSLPR polypeptide. As used herein,the terms, “specific” and “specificity” refer to the ability of theselective binding agents to bind to human TSLPR polypeptides and not tobind to human non-TSLPR polypeptides. It will be appreciated, however,that the selective binding agents may also bind orthologs of thepolypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8, that is, interspecies versions thereof, such as mouse and ratTSLPR polypeptides.

[0088] The term “transduction” is used to refer to the transfer of genesfrom one bacterium to another, usually by a phage. “Transduction” alsorefers to the acquisition and transfer of eukaryotic cellular sequencesby retroviruses.

[0089] The term “transfection” is used to refer to the uptake of foreignor exogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook etal., Molecular Cloning, A Laboratory Manual (Cold Spring HarborLaboratories, 1989); Davis et al., Basic Methods in Molecular Biology(Elsevier, 1986); and Chu et al., 1981, Gene 13:197. Such techniques canbe used to introduce one or more exogenous DNA moieties into suitablehost cells.

[0090] The term “transformation” as used herein refers to a change in acell's genetic characteristics, and a cell has been transformed when ithas been modified to contain a new DNA. For example, a cell istransformed where it is genetically modified from its native state.Following transfection or transduction, the transforming DNA mayrecombine with that of the cell by physically integrating into achromosome of the cell, may be maintained transiently as an episomalelement without being replicated, or may replicate independently as aplasmid. A cell is considered to have been stably transformed when theDNA is replicated with the division of the cell.

[0091] Relatedness of Nucleic Acid Molecules and/or Polypeptides

[0092] It is understood that related nucleic acid molecules includeallelic or splice variants of the nucleic acid molecule of any of SEQ IDNO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, andinclude sequences which are complementary to any of the above nucleotidesequences. Related nucleic acid molecules also include a nucleotidesequence encoding a polypeptide comprising or consisting essentially ofa substitution, modification, addition and/or deletion of one or moreamino acid residues compared to the polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. Such related TSLPRpolypeptides may comprise, for example, an addition and/or a deletion ofone or more N-linked or O-linked glycosylation sites or an additionand/or a deletion of one or more cysteine residues.

[0093] Related nucleic acid molecules also include fragments of TSLPRnucleic acid molecules which encode a polypeptide of at least about 25contiguous amino acids, or about 50 amino acids, or about 75 aminoacids, or about 100 amino acids, or about 150 amino acids, or about 200amino acids, or more than 200 amino acid residues of the TSLPRpolypeptide of any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.

[0094] In addition, related TSLPR nucleic acid molecules also includethose molecules which comprise nucleotide sequences which hybridizeunder moderately or highly stringent conditions as defined herein withthe fully complementary sequence of the TSLPR nucleic acid molecule ofany of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQID NO: 11, or of a molecule encoding a polypeptide, which polypeptidecomprises the amino acid sequence as shown in any of SEQ ID NO: 2, SEQID NO: 5, or SEQ ID NO: 8, or of a nucleic acid fragment as definedherein, or of a nucleic acid fragment encoding a polypeptide as definedherein. Hybridization probes may be prepared using the TSLPR sequencesprovided herein to screen cDNA, genomic or synthetic DNA libraries forrelated sequences. Regions of the DNA and/or amino acid sequence ofTSLPR polypeptide that exhibit significant identity to known sequencesare readily determined using sequence alignment algorithms as describedherein and those regions may be used to design probes for screening.

[0095] The term “highly stringent conditions” refers to those conditionsthat are designed to permit hybridization of DNA strands whose sequencesare highly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. See Sambrook, Fritsch & ManiatisMolecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: APractical Approach Ch. 4 (IRL Press Limited).

[0096] More stringent conditions (such as higher temperature, lowerionic strength, higher formamide, or other denaturing agent) may also beused—however, the rate of hybridization will be affected. Other agentsmay be included in the hybridization and washing buffers for the purposeof reducing non-specific and/or background hybridization. Examples are0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll,Denhardt's solution, sonicated salmon sperm DNA (or another noncomplementary DNA), and dextran sulfate, although other suitable agentscan also be used. The concentration and types of these additives can bechanged without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7.4; however, at typical ionic strength conditions, therate of hybridization is nearly independent of pH. See Anderson et al.,Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL PressLimited).

[0097] Factors affecting the stability of DNA duplex include basecomposition, length, and degree of base pair mismatch. Hybridizationconditions can be adjusted by one skilled in the art in order toaccommodate these variables and allow DNAs of different sequencerelatedness to form hybrids. The melting temperature of a perfectlymatched DNA duplex can be estimated by the following equation:

[0098] T_(m)(° C. )=81.5+16.6(log[Na+])+0.41(%G+C)−600/N−0.72(%formamide) where N is the length of the duplex formed, [Na+] is themolar concentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

[0099] The term “moderately stringent conditions” refers to conditionsunder which a DNA duplex with a greater degree of base pair mismatchingthan could occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By wayof example, “moderately stringent conditions” of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch.

[0100] It will be appreciated by those skilled in the art that there isno absolute distinction between “highly stringent conditions” and“moderately stringent conditions.” For example, at 0.015 M sodium ion(no formamide), the melting temperature of perfectly matched long DNA isabout 71° C. With a wash at 65° C. (at the same ionic strength), thiswould allow for approximately a 6% mismatch. To capture more distantlyrelated sequences, one skilled in the art can simply lower thetemperature or raise the ionic strength.

[0101] A good estimate of the melting temperature in IM NaCl* foroligonucleotide probes up to about 20 nt is given by:

[0102] Tm=2° C. per A-T base pair+4° C. per G-C base pair

[0103] *The sodium ion concentration in 6X salt sodium citrate (SSC) isIM. See Suggs et al., Developmental Biology Using Purified Genes 683(Brown and Fox, eds., 1981).

[0104] High stringency washing conditions for oligonucleotides areusually at a temperature of 0-5° C. below the Tm of the oligonucleotidein 6X SSC, 0.1% SDS.

[0105] In another embodiment, related nucleic acid molecules comprise orconsist of a nucleotide sequence that is at least about 70 percentidentical to the nucleotide sequence as shown in any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, or compriseor consist essentially of a nucleotide sequence encoding a polypeptidethat is at least about 70 percent identical to the polypeptide as setforth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. Inpreferred embodiments, the nucleotide sequences are about 75 percent, orabout 80 percent, or about 85 percent, or about 90 percent, or about 95,96, 97, 98, or 99 percent identical to the nucleotide sequence as shownin any of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, orSEQ ID NO: 11, or the nucleotide sequences encode a polypeptide that isabout 75 percent, or about 80 percent, or about 85 percent, or about 90percent, or about 95, 96, 97, 98, or 99 percent identical to thepolypeptide sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5,or SEQ ID NO: 8. Related nucleic acid molecules encode polypeptidespossessing at least one activity of the polypeptide set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.

[0106] Differences in the nucleic acid sequence may result inconservative and/or non-conservative modifications of the amino acidsequence relative to the amino acid sequence of any of SEQ ID NO: 2, SEQID NO: 5, or SEQ ID NO: 8.

[0107] Conservative modifications to the amino acid sequence of any ofSEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 (and the correspondingmodifications to the encoding nucleotides) will produce a polypeptidehaving functional and chemical characteristics similar to those of TSLPRpolypeptides. In contrast, substantial modifications in the functionaland/or chemical characteristics of TSLPR polypeptides may beaccomplished by selecting substitutions in the amino acid sequence ofany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 that differsignificantly in their effect on maintaining (a) the structure of themolecular backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

[0108] For example, a “conservative amino acid substitution” may involvea substitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

[0109] Conservative amino acid substitutions also encompassnon-naturally occurring amino acid residues that are typicallyincorporated by chemical peptide synthesis rather than by synthesis inbiological systems. These include peptidomimetics, and other reversed orinverted forms of amino acid moieties.

[0110] Naturally occurring residues may be divided into classes based oncommon side chain properties:

[0111] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0112] 2) neutral hydrophilic: Cys, Ser, Thr;

[0113] 3) acidic: Asp, Glu;

[0114] 4) basic: Asn, Gln, His, Lys, Arg;

[0115] 5) residues that influence chain orientation: Gly, Pro; and

[0116] 6) aromatic: Trp, Tyr, Phe.

[0117] For example, non-conservative substitutions may involve theexchange of a member of one of these classes for a member from anotherclass. Such substituted residues may be introduced into regions of thehuman TSLPR polypeptide that are homologous with non-human TSLPRpolypeptides, or into the non-homologous regions of the molecule.

[0118] In making such changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics. Thehydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0119] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0120] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

[0121] The following hydrophilicity values have been assigned to theseamino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. One may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

[0122] Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the TSLPRpolypeptide, or to increase or decrease the affinity of the TSLPRpolypeptides described herein. Exemplary amino acid substitutions areset forth in Table I. Amino Acid Substitutions Original ResiduesExemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile ValArg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln AsnAsn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu,Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met,Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe,Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, CysThr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile,Met, Leu, Phe, Leu Ala, Norleucine

[0123] A skilled artisan will be able to determine suitable variants ofthe polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, orSEQ ID NO: 8 using well known techniques. For identifying suitable areasof the molecule that may be changed without destroying biologicalactivity, one skilled in the art may target areas not believed to beimportant for activity. For example, when similar polypeptides withsimilar activities from the same species or from other species areknown, one skilled in the art may compare the amino acid sequence of aTSLPR polypeptide to such similar polypeptides. With such a comparison,one can identify residues and portions of the molecules that areconserved among similar polypeptides. It will be appreciated thatchanges in areas of the TSLPR molecule that are not conserved relativeto such similar polypeptides would be less likely to adversely affectthe biological activity and/or structure of a TSLPR polypeptide. Oneskilled in the art would also know that, even in relatively conservedregions, one may substitute chemically similar amino acids for thenaturally occurring residues while retaining activity (conservativeamino acid residue substitutions). Therefore, even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

[0124] Additionally, one skilled in the art can reviewstructure-function studies identifying residues in similar polypeptidesthat are important for activity or structure. In view of such acomparison, one can predict the importance of amino acid residues in aTSLPR polypeptide that correspond to amino acid residues that areimportant for activity or structure in similar polypeptides. One skilledin the art may opt for chemically similar amino acid substitutions forsuch predicted important amino acid residues of TSLPR polypeptides.

[0125] One skilled in the art can also analyze the three-dimensionalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of such information, one skilled in theart may predict the alignment of amino acid residues of TSLPRpolypeptide with respect to its three dimensional structure. One skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each amino acid residue. The variants couldbe screened using activity assays known to those with skill in the art.Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change would be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

[0126] A number of scientific publications have been devoted to theprediction of secondary structure. See Moult, 1996, Curr. Opin.Biotechnol. 7:422-27; Chou et al., 1974, Biochemistry 13:222-45; Chou etal., 1974, Biochemistry 113:211-22; Chou et al., 1978, Adv. Enzymol.Relat. Areas Mol. Biol. 47:45-48; Chou et al., 1978, Ann. Rev. Biochem.47:251-276; and Chou et al., 1979, Biophys. J. 26:367-84. Moreover,computer programs are currently available to assist with predictingsecondary structure. One method of predicting secondary structure isbased upon homology modeling. For example, two polypeptides or proteinswhich have a sequence identity of greater than 30%, or similaritygreater than 40%, often have similar structural topologies. The recentgrowth of the protein structural database (PDB) has provided enhancedpredictability of secondary structure, including the potential number offolds within the structure of a polypeptide or protein. See Holm et al.,1999, Nucleic Acids Res. 27:244-47. It has been suggested that there area limited number of folds in a given polypeptide or protein and thatonce a critical number of structures have been resolved, structuralprediction will become dramatically more accurate (Brenner et al., 1997,Curr. Opin. Struct. Biol. 7:369-76).

[0127] Additional methods of predicting secondary structure include“threading” (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl etal., 1996, Structure 4:15-19), “profile analysis” (Bowie et al, 1991,Science, 253:164-70; Gribskov et al., 1990,Methods Enzymol. 183:146-59;Gribskov et al., 1987, Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and“evolutionary linkage” (See Holm et al., supra, and Brenner et al.,supra).

[0128] Preferred TSLPR polypeptide variants include glycosylationvariants wherein the number and/or type of glycosylation sites have beenaltered compared to the amino acid sequence set forth in any of SEQ IDNO 2, SEQ ID NO: 5, or SEQ ID NO: 8. In one embodiment, TSLPRpolypeptide variants comprise a greater or a lesser number of N-linkedglycosylation sites than the amino acid sequence set forth in any of SEQID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. An N-linked glycosylation siteis characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein theamino acid residue designated as X may be any amino acid residue exceptproline. The substitution of amino acid residues to create this sequenceprovides a potential new site for the addition of an N-linkedcarbohydrate chain. Alternatively, substitutions that eliminate thissequence will remove an existing N-linked carbohydrate chain. Alsoprovided is a rearrangement of N-linked carbohydrate chains wherein oneor more N-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated. Additional preferred TSLPR variants include cysteine variants,wherein one or more cysteine residues are deleted or substituted withanother amino acid (e.g., serine) as compared to the amino acid sequenceset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.Cysteine variants are useful when TSLPR polypeptides must be refoldedinto a biologically active conformation such as after the isolation ofinsoluble inclusion bodies. Cysteine variants generally have fewercysteine residues than the native protein, and typically have an evennumber to minimize interactions resulting from unpaired cysteines.

[0129] In other embodiments, related nucleic acid molecules comprise orconsist of a nucleotide sequence encoding a polypeptide as set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least oneamino acid insertion and wherein the polypeptide has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8, or a nucleotide sequence encoding a polypeptide as set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8with at least oneamino acid deletion and wherein the polypeptide has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ IDNO: 8. Related nucleic acid molecules also comprise or consist of anucleotide sequence encoding a polypeptide as set forth in any of SEQ IDNO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 wherein the polypeptide has acarboxyl- and/or amino-terminal truncation and further wherein thepolypeptide has an activity of the polypeptide set forth in any of SEQID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. Related nucleic acid moleculesalso comprise or consist of a nucleotide sequence encoding a polypeptideas set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 withat least one modification selected from the group consisting of aminoacid substitutions, amino acid insertions, amino acid deletions,carboxyl-terminal truncations, and amino-terminal truncations andwherein the polypeptide has an activity of the polypeptide set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.

[0130] In addition, the polypeptide comprising the amino acid sequenceof any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or other TSLPRpolypeptide, may be fused to a homologous polypeptide to form ahomodimer or to a heterologous polypeptide to form a heterodimer.Heterologous peptides and polypeptides include, but are not limited to:an epitope to allow for the detection and/or isolation of a TSLPR fusionpolypeptide; a transmembrane receptor protein or a portion thereof, suchas an extracellular domain or a transmembrane and intracellular domain;a ligand or a portion thereof which binds to a transmembrane receptorprotein; an enzyme or portion thereof which is catalytically active; apolypeptide or peptide which promotes oligomerization, such as a leucinezipper domain; a polypeptide or peptide which increases stability, suchas an immunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide comprising the aminoacid sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQID NO: 8, or other TSLPR polypeptide.

[0131] Fusions can be made either at the amino-terminus or at thecarboxyl-terminus of the polypeptide comprising the amino acid sequenceset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, orother TSLPR polypeptide. Fusions may be direct with no linker or adaptermolecule or may be through a linker or adapter molecule. A linker oradapter molecule may be one or more amino acid residues, typically fromabout 20 to about 50 amino acid residues. A linker or adapter moleculemay also be designed with a cleavage site for a DNA restrictionendonuclease or for a protease to allow for the separation of the fusedmoieties. It will be appreciated that once constructed, the fusionpolypeptides can be derivatized according to the methods describedherein.

[0132] In a further embodiment of the invention, the polypeptidecomprising the amino acid sequence of any of SEQ ID NO: 2, SEQ ID NO: 5,or SEQ ID NO: 8, or other TSLPR polypeptide, is fused to one or moredomains of an Fe region of human IgG. Antibodies comprise twofunctionally independent parts, a variable domain known as “Fab,” thatbinds an antigen, and a constant domain known as “Fc,” that is involvedin effector functions such as complement activation and attack byphagocytic cells. An Fe has a long serum half-life, whereas an Fab isshort-lived. Capon et al., 1989, Nature 337:525-31. When constructedtogether with a therapeutic protein, an Fc domain can provide longerhalf-life or incorporate such functions as Fe receptor binding, proteinA binding, complement fixation, and perhaps even placental transfer. Id.Table II summarizes the use of certain Fc fusions known in the art.TABLE II Fc Fusion with Therapeutic Proteins Therapeutic Form of FcFusion partner implications Reference IgG1 N-terminus of Hodgkin's U.S.Pat. No. CD30-L disease; 5,480,981 anaplastic lymphoma; T-cell leukemiaMurine IL-10 anti- Zheng et al., 1995, J. Fcγ2a inflammatory; Immunol.154:5590-600 transplant rejection IgG1 TNF receptor septic shock Fisheret al., 1996, N. Engl. J. Med. 334:1697- 1702; Van Zee et al., 1996, J.Immunol. 156:2221-30 IgG, IgA, TNF receptor inflammation, U.S. Pat. No.IgM, or IgE autoimmune 5,808,029 (excluding disorders the first domain)IgG1 CD4 receptor AIDS Capon et al., 1989, Nature 337: 525-31 IgG1,N-terminus anti-cancer, Harvill et al., 1995, IgG3 of IL-2 antiviralImmunotech. 1:95-105 IgG1 C-terminus of osteoarthritis; WO 97/23614 OPGbone density IgG1 N-terminus of anti-obesity PCT/US 97/23183, filedleptin December 11, 1997 Human CTLA-4 autoimmune Linsley, 1991, J. Exp.Ig Cγ1 disorders Med., 174:561-69

[0133] In one example, a human IgG hinge, CH2, and CH3 region may befused at either the amino-terminus or carboxyl-terminus of the TSLPRpolypeptides using methods known to the skilled artisan. In anotherexample, a human IgG hinge, CH2, and CH3 region may be fused at eitherthe amino-terminus or carboxyl-terminus of a TSLPR polypeptide fragment(e.g., the predicted extracellular portion of TSLPR polypeptide).

[0134] The resulting TSLPR fusion polypeptide may be purified by use ofa Protein A affinity column. Peptides and proteins fused to an Fc regionhave been found to exhibit a substantially greater half-life in vivothan the unfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be a naturally occurring Fc region, or may be altered to improvecertain qualities, such as therapeutic qualities, circulation time, orreduced aggregation.

[0135] Identity and similarity of related nucleic acid molecules andpolypeptides are readily calculated by known methods. Such methodsinclude, but are not limited to those described in ComputationalMolecular Biology (A. M. Lesk, ed., Oxford University Press 1988);Biocomputing: Informatics and Genome Projects (D. W. Smith, ed.,Academic Press 1993); Computer Analysis of Sequence Data (Part 1, A. M.Griffin and H. G. Griffin, eds., Humana Press 1994); G. vonHeinle,Sequence Analysis in Molecular Biology (Academic Press 1987);Sequence Analysis Primer (M. Gribskov and J. Devereux, eds., M. StocktonPress 1991); and Carillo et al., 1988, SIAM J. Applied Math., 48:1073.

[0136] Preferred methods to determine identity and/or similarity aredesigned to give the largest match between the sequences tested. Methodsto determine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,1990, J. Mol. Biol. 215:403-10). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda,Md.); Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

[0137] Certain alignment schemes for aligning two amino acid sequencesmay result in the matching of only a short region of the two sequences,and this small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the claimed polypeptide.

[0138] For example, using the computer algorithm GAP (Genetics ComputerGroup, University of Wisconsin, Madison, Wis.), two polypeptides forwhich the percent sequence identity is to be determined are aligned foroptimal matching of their respective amino acids (the “matched span,” asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3×the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 0.1×the gap opening penalty), as well as a comparison matrixsuch as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.A standard comparison matrix is also used by the algorithm (see Dayhoffet al., 5 Atlas of Protein Sequence and Structure (Supp. 31978) (PAM250comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci USA89:10915-19 (BLOSUM 62 comparison matrix)).

[0139] Preferred parameters for polypeptide sequence comparison includethe following:

[0140] Algorithm: Needleman and Wunsch, 1970,J. Mol. Biol. 48:443-53;

[0141] Comparison matrix: BLOSUM 62 (Henikoff et al., supra);

[0142] Gap Penalty: 12

[0143] Gap Length Penalty: 4

[0144] Threshold of Similarity: 0

[0145] The GAP program is useful with the above parameters. Theaforementioned parameters are the default parameters for polypeptidecomparisons (along with no penalty for end gaps) using the GAPalgorithm.

[0146] Preferred parameters for nucleic acid molecule sequencecomparison include the following:

[0147] Algorithm: Needleman and Wunsch, supra;

[0148] Comparison matrix: matches=+10, mismatch=0

[0149] Gap Penalty: 50

[0150] Gap Length Penalty: 3

[0151] The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

[0152] Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, and thresholds of similarity may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

[0153] Nucleic Acid Molecules

[0154] The nucleic acid molecules encoding a polypeptide comprising theamino acid sequence of a TSLPR polypeptide can readily be obtained in avariety of ways including, without limitation, chemical synthesis, cDNAor genomic library screening, expression library screening, and/or PCRamplification of cDNA.

[0155] Recombinant DNA methods used herein are generally those set forthin Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) and/or Current Protocols in MolecularBiology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons1994). The invention provides for nucleic acid molecules as describedherein and methods for obtaining such molecules.

[0156] Where a gene encoding the amino acid sequence of a TSLPRpolypeptide has been identified from one species, all or a portion ofthat gene maybe used as a probe to identify orthologs or related genesfrom the same species. The probes or primers may be used to screen cDNAlibraries from various tissue sources believed to express the TSLPRpolypeptide. In addition, part or all of a nucleic acid molecule havingthe sequence as set forth in any of SEQ ID NO: 1, SEQ ID NO: 4, SEQ IDNO: 7, SEQ ID NO: 10, or SEQ ID NO: 11 may be used to screen a genomiclibrary to identify and isolate a gene encoding the amino acid sequenceof a TSLPR polypeptide. Typically, conditions of moderate or highstringency will be employed for screening to minimize the number offalse positives obtained from the screening.

[0157] Nucleic acid molecules encoding the amino acid sequence of TSLPRpolypeptides may also be identified by expression cloning which employsthe detection of positive clones based upon a property of the expressedprotein. Typically, nucleic acid libraries are screened by the bindingan antibody or other binding partner (e.g., receptor or ligand) tocloned proteins that are expressed and displayed on a host cell surface.The antibody or binding partner is modified with a detectable label toidentify those cells expressing the desired clone.

[0158] Recombinant expression techniques conducted in accordance withthe descriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence that encodes the amino acid sequenceof a TSLPR polypeptide into an appropriate vector, one skilled in theart can readily produce large quantities of the desired nucleotidesequence. The sequences can then be used to generate detection probes oramplification primers. Alternatively, a polynucleotide encoding theamino acid sequence of a TSLPR polypeptide can be inserted into anexpression vector. By introducing the expression vector into anappropriate host, the encoded TSLPR polypeptide may be produced in largeamounts.

[0159] Another method for obtaining a suitable nucleic acid sequence isthe polymerase chain reaction (PCR). In this method, cDNA is preparedfrom poly(A)+RNA or total RNA using the enzyme reverse transcriptase.Two primers, typically complementary to two separate regions of cDNAencoding the amino acid sequence of a TSLPR polypeptide, are then addedto the cDNA along with a polymerase such as Taq polymerase, and thepolymerase amplifies the cDNA region between the two primers.

[0160] Another means of preparing a nucleic acid molecule encoding theamino acid sequence of a TSLPR polypeptide is chemical synthesis usingmethods well known to the skilled artisan such as those described byEngels et al., 1989, Angew. Chem. Intl. Ed. 28:716-34. These methodsinclude, inter alia, the phosphotriester, phosphoramidite, andH-phosphonate methods for nucleic acid synthesis. A preferred method forsuch chemical synthesis is polymer-supported synthesis using standardphosphoramidite chemistry. Typically, the DNA encoding the amino acidsequence of a TSLPR polypeptide will be several hundred nucleotides inlength. Nucleic acids larger than about 100 nucleotides can besynthesized as several fragments using these methods. The fragments canthen be ligated together to form the full-length nucleotide sequence ofa TSLPR gene. Usually, the DNA fragment encoding the amino-terminus ofthe polypeptide will have an ATG, which encodes a methionine residue.This methionine may or may not be present on the mature form of theTSLPR polypeptide, depending on whether the polypeptide produced in thehost cell is designed to be secreted from that cell. Other methods knownto the skilled artisan may be used as well.

[0161] In certain embodiments, nucleic acid variants contain codonswhich have been altered for optimal expression of a TSLPR polypeptide ina given host cell. Particular codon alterations will depend upon theTSLPR polypeptide and host cell selected for expression. Such “codonoptimization” can be carried out by a variety of methods, for example,by selecting codons which are preferred for use in highly expressedgenes in a given host cell. Computer algorithms which incorporate codonfrequency tables such as “Eco_high.Cod” for codon preference of highlyexpressed bacterial genes may be used and are provided by the Universityof Wisconsin Package Version 9.0 (Genetics Computer Group, Madison,Wis.). Other useful codon frequency tables include “Celegans_high.cod,”“Celegans_low.cod,” “Drosophila_high.cod,” “Human_high.cod,”“Maize_high.cod,” and “Yeast_high.cod.”

[0162] In some cases, it may be desirable to prepare nucleic acidmolecules encoding TSLPR polypeptide variants. Nucleic acid moleculesencoding variants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

[0163] Vectors and Host Cells

[0164] A nucleic acid molecule encoding the amino acid sequence of aTSLPR polypeptide is inserted into an appropriate expression vectorusing standard ligation techniques. The vector is typically selected tobe functional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding the amino acid sequence of a TSLPR polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether a TSLPR polypeptide is to be post-translationallymodified (e.g., glycosylated and/or phosphorylated). If so, yeast,insect, or mammalian host cells are preferable. For a review ofexpression vectors, see Meth. Enz., vol. 185 (D. V. Goeddel, ed.,Academic Press 1990).

[0165] Typically, expression vectors used in any of the host cells willcontain sequences for plasmid maintenance and for cloning and expressionof exogenous nucleotide sequences. Such sequences, collectively referredto as “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

[0166] Optionally, the vector may contain a “tag”-encoding sequence,i.e., an oligonucleotide molecule located at the 5′ or 3′ end of theTSLPR polypeptide coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification of the TSLPR polypeptide from the host cell. Affinitypurification can be accomplished, for example, by column chromatographyusing antibodies against the tag as an affinity matrix. Optionally, thetag can subsequently be removed from the purified TSLPR polypeptide byvarious means such as using certain peptidases for cleavage.

[0167] Flanking sequences may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof flanking sequences from more than one source), or synthetic, or theflanking sequences may be native sequences which normally function toregulate TSLPR polypeptide expression. As such, the source of a flankingsequence may be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the flankingsequence is functional in, and can be activated by, the host cellmachinery.

[0168] Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein—other than the TSLPR gene flankingsequences—will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

[0169] Where all or only a portion of the flanking sequence is known, itmay be obtained using PCR and/or by screening a genomic library with asuitable oligonucleotide and/or flanking sequence fragment from the sameor another species. Where the flanking sequence is not known, a fragmentof DNA containing a flanking sequence may be isolated from a largerpiece of DNA that may contain, for example, a coding sequence or evenanother gene or genes. Isolation may be accomplished by restrictionendonuclease digestion to produce the proper DNA fragment followed byisolation using agarose gel purification, Qiagen® column chromatography(Chatsworth, Calif.), or other methods known to the skilled artisan. Theselection of suitable enzymes to accomplish this purpose will be readilyapparent to one of ordinary skill in the art.

[0170] An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of a TSLPR polypeptide. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria andvarious origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitusvirus (VSV), or papillomaviruses such as HPV or BPV) are useful forcloning vectors in mammalian cells. Generally, the origin of replicationcomponent is not needed for mammalian expression vectors (for example,the SV40 origin is often used only because it contains the earlypromoter).

[0171] A transcription termination sequence is typically located 3′ ofthe end of a polypeptide coding region and serves to terminatetranscription. Usually, a transcription termination sequence inprokaryotic cells is a G-C rich fragment followed by a poly-T sequence.While the sequence is easily cloned from a library or even purchasedcommercially as part of a vector, it can also be readily synthesizedusing methods for nucleic acid synthesis such as those described herein.

[0172] A selectable marker gene element encodes a protein necessary forthe survival and growth of a host cell grown in a selective culturemedium. Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells; (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

[0173] Other selection genes may be used to amplify the gene that willbe expressed. Amplification is the process wherein genes that are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whereinonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes a TSLPR polypeptide. As a result, increased quantitiesof TSLPR polypeptide are synthesized from the amplified DNA.

[0174] A ribosome binding site is usually necessary for translationinitiation of mRNA and is characterized by a Shine-Dalgamo sequence(prokaryotes) or a Kozak sequence (eukaryotes). The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of a TSLPRpolypeptide to be expressed. The ShineDalgamo sequence is varied but istypically a polypurine (i.e., having a high A-G content). ManyShine-Dalgamo sequences have been identified, each of which can bereadily synthesized using methods set forth herein and used in aprokaryotic vector.

[0175] A leader, or signal, sequence may be used to direct a TSLPRpolypeptide out of the host cell. Typically, a nucleotide sequenceencoding the signal sequence is positioned in the coding region of aTSLPR nucleic acid molecule, or directly at the 5′ end of a TSLPRpolypeptide coding region. Many signal sequences have been identified,and any of those that are functional in the selected host cell may beused in conjunction with a TSLPR nucleic acid molecule. Therefore, asignal sequence may be homologous (naturally occurring) or heterologousto the TSLPR nucleic acid molecule. Additionally, a signal sequence maybe chemically synthesized using methods described herein. In most cases,the secretion of a TSLPR polypeptide from the host cell via the presenceof a signal peptide will result in the removal of the signal peptidefrom the secreted TSLPR polypeptide. The signal sequence may be acomponent of the vector, or it may be a part of a TSLPR nucleic acidmolecule that is inserted into the vector.

[0176] Included within the scope of this invention is the use of eithera nucleotide sequence encoding a native TSLPR polypeptide signalsequence joined to a TSLPR polypeptide coding region or a nucleotidesequence encoding a heterologous signal sequence joined to a TSLPRpolypeptide coding region. The heterologous signal sequence selectedshould be one that is recognized and processed, i.e., cleaved by asignal peptidase, by the host cell. For prokaryotic host cells that donot recognize and process the native TSLPR polypeptide signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, or heat-stable enterotoxin II leaders. For yeastsecretion, the native TSLPR polypeptide signal sequence may besubstituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian TSLPR polysignal sequences may besuitable.

[0177] In some cases, such as where glycosylation is desired in aeukaryotic host cell expression system, one may manipulate the variouspresequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addpro-sequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired TSLPR polypeptide, if the enzymecuts at such area within the mature polypeptide.

[0178] In many cases, transcription of a nucleic acid molecule isincreased by the presence of one or more introns in the vector; this isparticularly true where a polypeptide is produced in eukaryotic hostcells, especially mammalian host cells. The introns used may benaturally occurring within the TSLPR gene especially where the gene usedis a full-length genomic sequence or a fragment thereof. Where theintron is not naturally occurring within the gene (as for most cDNAs),the intron may be obtained from another source. The position of theintron with respect to flanking sequences and the TSLPR gene isgenerally important, as the intron must be transcribed to be effective.Thus, when a TSLPR cDNA molecule is being transcribed, the preferredposition for the intron is 3′ to the transcription start site and 5′ tothe poly-A transcription termination sequence. Preferably, the intron orintrons will be located on one side or the other (i.e., 5′ or 3′) of thecDNA such that it does not interrupt the coding sequence. Any intronfrom any source, including viral, prokaryotic and eukaryotic (plant oranimal) organisms, may be used to practice this invention, provided thatit is compatible with the host cell into which it is inserted. Alsoincluded herein are synthetic introns. Optionally, more than one intronmay be used in the vector.

[0179] The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the TSLPR polypeptide.Promoters are untranscribed sequences located upstream (i.e., 5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control the transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno control over gene expression. A large number of promoters, recognizedby a variety of potential host cells, are well known. A suitablepromoter is operably linked to the DNA encoding TSLPR polypeptide byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.The native TSLPR promoter sequence may be used to direct amplificationand/or expression of a TSLPR nucleic acid molecule. A heterologouspromoter is preferred, however, if it permits greater transcription andhigher yields of the expressed protein as compared to the nativepromoter, and if it is compatible with the host cell system that hasbeen selected for use.

[0180] Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase; atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence, using linkers or adapters asneeded to supply any useful restriction sites.

[0181] Suitable promoters for use with yeast hosts are also well knownin the art. Yeast enhancers are advantageously used with yeastpromoters. Suitable promoters for use with mammalian host cells are wellknown and include, but are not limited to, those obtained from thegenomes of viruses such as polyoma virus, fowlpox virus, adenovirus(such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

[0182] Additional promoters which may be of interest in controllingTSLPR gene expression include, but are not limited to: the SV40 earlypromoter region (Bemoist and Chambon, 1981, Nature 290:304-10); the CMVpromoter; the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1444-45); the regulatory sequences of the metallothionine gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the beta-lactamase promoter (Villa-Kamaroff et al.,1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter(DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Also ofinterest are the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:the elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987,Hepatology 7:425-515); the insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); theimmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature318:533-38; Alexander et al., 1987, Mol. Cell. Biol., 7:1436-44); themouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95);the albumin gene control region which is active in liver (Pinkert etal., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein genecontrol region which is active in liver (Krumlauf et al., 1985, Mol.Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); thealpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-71); the beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, 1985, Nature 314:283-86); and the gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science234:1372-78).

[0183] An enhancer sequence may be inserted into the vector to increasethe transcription of a DNA encoding a TSLPR polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus TSLPR polyenhancers are exemplary enhancingelements for the activation of eukaryotic promoters. While an enhancermay be spliced into the vectorat a position 5′ or 3′ to a TSLPR nucleicacid molecule, it is typically located at a site 5′ from the promoter.

[0184] Expression vectors of the invention may be constructed from astarting vector such as a commercially available vector. Such vectorsmay or may not contain all of the desired flanking sequences. Where oneor more of the flanking sequences described herein are not alreadypresent in the vector, they may be individually obtained and ligatedinto the vector. Methods used for obtaining each of the flankingsequences are well known to one skilled in the art.

[0185] Preferred vectors for practicing this invention are those whichare compatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen, SanDiego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen,Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2(Clontech, Palo Alto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha(PCT Pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island,N.Y.).

[0186] Additional suitable vectors include, but are not limited to,cosmids, plasmids, or modified viruses, but it will be appreciated thatthe vector system must be compatible with the selected host cell. Suchvectors include, but are not limited to plasmids such as Bluescriptplasmid derivatives (a high copy number ColE1-based phagemid; StratageneCloning Systems, La Jolla Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit andPC2.1® plasmid derivatives; Invitrogen), and mammalian, yeast or virusvectors such as a baculovirus expression system (pBacPAK plasmidderivatives; Clontech).

[0187] After the vector has been constructed and a nucleic acid moleculeencoding a TSLPR polypeptide has been inserted into the proper site ofthe vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for a TSLPR polypeptide into a selected hostcell may be accomplished by well known methods including methods such astransfection, infection, calcium chloride, electroporation,microinjection, lipofection, DEAE-dextran method, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., supra.

[0188] Host cells may be prokaryotic host cells (such as E. coli) oreukaryotic host cells (such as a yeast, insect, or vertebrate cell). Thehost cell, when cultured under appropriate conditions, synthesizes aTSLPR polypeptide which can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule.

[0189] A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), Manassas,Va. Examples include, but are not limited to, mammalian cells, such asChinese hamster ovary cells (CHO), CHO DHFR(−) cells (Urlaub et al.,1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20), human embryonic kidney(HEK) 293 or 293T cells, or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening, product production, and purification are knownin the art.

[0190] Other suitable mammalian cell lines, are the monkey COS-1 andCOS-7 cell lines, and the CV-1 cell line. Further exemplary mammalianhost cells include primate cell lines and rodent cell lines, includingtransformed cell lines. Normal diploid cells, cell strains derived fromin vitro culture of primary tissue, as well as primary explants, arealso suitable. Candidate cells may be genotypically deficient in theselection gene, or may contain a dominantly acting selection gene. Othersuitable mammalian cell lines include but are not limited to, mouseneuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived fromSwiss, Balb-c or NIH mice, BHK or HaK hamster cell lines. Each of thesecell lines is known by and available to those skilled in the art ofprotein expression.

[0191] Similarly useful as host cells suitable for the present inventionare bacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α, DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

[0192] Many strains of yeast cells known to those skilled in the art arealso available as host cells for the expression of the polypeptides ofthe present invention. Preferred yeast cells include, for example,Saccharomyces cerivisae and Pichia pastoris.

[0193] Additionally, where desired, insect cell systems may be utilizedin the methods of the present invention. Such systems are described, forexample, in Kitts et al., 1993, Biotechniques, 14:810-17; Lucklow, 1993,Curr. Opin. Biotechnol. 4:564-72; and Lucklow et al., 1993, J. Virol.,67:4566-79. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

[0194] One may also use transgenic animals to express glycosylated TSLPRpolypeptides. For example, one may use a transgenic milk-producinganimal (a cow or goat, for example) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produce TSLPRpolypeptides, however, in general, the glycosylation occurring in plantsis different from that produced in mammalian cells, and may result in aglycosylated product which is not suitable for human therapeutic use.

[0195] Polypeptide Production

[0196] Host cells comprising a TSLPR polypeptide expression vector maybe cultured using standard media well known to the skilled artisan. Themedia will usually contain all nutrients necessary for the growth andsurvival of the cells. Suitable media for culturing E. coli cellsinclude, for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells include Roswell ParkMemorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium(MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which maybe supplemented with serum and/or growth factors as necessary for theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

[0197] Typically, an antibiotic or other compound useful for selectivegrowth of transfected or transformed cells is added as a supplement tothe media. The compound to be used will be dictated by the selectablemarker element present on the plasmid with which the host cell wastransformed. For example, where the selectable marker element iskanamycin resistance, the compound added to the culture medium will bekanamycin. Other compounds for selective growth include ampicillin,tetracycline, and neomycin.

[0198] The amount of a TSLPR polypeptide produced by a host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, High PerformanceLiquid Chromatography (HPLC) separation, immunoprecipitation, and/oractivity assays such as DNA binding gel shift assays.

[0199] If a TSLPR polypeptide has been designed to be secreted from thehost cells, the majority of polypeptide may be found in the cell culturemedium. If however, the TSLPR polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram-negative bacteriahost cells).

[0200] For a TSLPR polypeptide situated in the host cell cytoplasmand/or nucleus (for eukaryotic host cells) or in the cytosol (forbacterial host cells), the intracellular material (including inclusionbodies for gram-negative bacteria) can be extracted from the host cellusing any standard technique known to the skilled artisan. For example,the host cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

[0201] If a TSLPR polypeptide has formed inclusion bodies in thecytosol, the inclusion bodies can often bind to the inner and/or outercellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material can then be treatedat pH extremes or with a chaotropic agent such as a detergent,guanidine, guanidine derivatives, urea, or urea derivatives in thepresence of a reducing agent such as dithiothreitol at alkaline pH ortris carboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The solubilized TSLPR polypeptide canthen be analyzed using gel electrophoresis, immunoprecipitation, or thelike. If it is desired to isolate the TSLPR polypeptide, isolation maybe accomplished using standard methods such as those described hereinand in Marston et al., 1990, Meth. Enz., 182:264-75.

[0202] In some cases, a TSLPR polypeptide may not be biologically activeupon isolation. Various methods for “refolding” or converting thepolypeptide to its tertiary structure and generating disulfide linkagescan be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridges. Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-2-mercaptoethanol(bME)/dithio-b(ME). In many instances, a cosolventmay be used or may be needed to increase the efficiency of therefolding, and the more common reagents used for this purpose includeglycerol, polyethylene glycol of various molecular weights, arginine andthe like.

[0203] If inclusion bodies are not formed to a significant degree uponexpression of a TSLPR polypeptide, then the polypeptide will be foundprimarily in the supernatant after centrifugation of the cellhomogenate. The polypeptide may be further isolated from the supernatantusing methods such as those described herein.

[0204] The purification of a TSLPR polypeptide from solution can beaccomplished using a variety of techniques. If the polypeptide has beensynthesized such that it contains a tag such as Hexahistidine (TSLPRpolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen) at either its carboxyl- oramino-terminus, it may be purified in a one-step process by passing thesolution through an affinity column where the column matrix has a highaffinity for the tag.

[0205] For example, polyhistidine binds with great affinity andspecificity to nickel. Thus, an affinity column of nickel (such as theQiagen® nickel columns) can be used for purification of TSLPRpolypeptide/polyHis. See, e.g., Current Protocols in Molecular Biology §10.11.8 (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons1993).

[0206] Additionally, TSLPR polypeptides may be purified through the useof a monoclonal antibody that is capable of specifically recognizing andbinding to a TSLPR polypeptide.

[0207] Other suitable procedures for purification include, withoutlimitation, affinity chromatography, immunoaffinity chromatography, ionexchange chromatography, molecular sieve chromatography, HPLC,electrophoresis (including native gel electrophoresis) followed by gelelution, and preparative isoelectric focusing (“Isoprime”machine/technique, Hoefer Scientific, San Francisco, Calif.). In somecases, two or more purification techniques may be combined to achieveincreased purity.

[0208] TSLPR polypeptides may also be prepared by chemical synthesismethods (such as solid phase peptide synthesis) using techniques knownin the art such as those set forth by Merrifield et al., 1963, J. Am.Chem. Soc. 85:2149; Houghten et al., 1985, Proc Natl Acad. Sci. USA82:5132; and Stewart and Young, Solid Phase Peptide Synthesis (PierceChemical Co. 1984). Such polypeptides may be synthesized with or withouta methionine on the amino-terminus. Chemically synthesized TSLPRpolypeptides may be oxidized using methods set forth in these referencesto form disulfide bridges. Chemically synthesized TSLPR polypeptides areexpected to have comparable biological activity to the correspondingTSLPR polypeptides produced recombinantly or purified from naturalsources, and thus may be used interchangeably with a recombinant ornatural TSLPR polypeptide.

[0209] Another means of obtaining TSLPR polypeptide is via purificationfrom biological samples such as source tissues and/or fluids in whichthe TSLPR polypeptide is naturally found. Such purification can beconducted using methods for protein purification as described herein.The presence of the TSLPR polypeptide during purification may bemonitored, for example, using an antibody prepared against recombinantlyproduced TSLPR polypeptide or peptide fragments thereof.

[0210] A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and the methods can be used toproduce polypeptides having specificity for TSLPR polypeptide. See,e.g., Roberts et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:12297-303,which describes the production of fusion proteins between an mRNA andits encoded peptide. See also, Roberts, 1999, Curr. Opin. Chem. Biol.3:268-73. Additionally, U.S. Pat. No. 5,824,469 describes methods forobtaining oligonucleotides capable of carrying out a specific biologicalfunction. The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those that exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated.

[0211] U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483describe processes for producing peptides or polypeptides. This is doneby producing stochastic genes or fragments thereof, and then introducingthese genes into host cells which produce one or more proteins encodedby the stochastic genes. The host cells are then screened to identifythose clones producing peptides or polypeptides having the desiredactivity.

[0212] Another method for producing peptides or polypeptides isdescribed in PCT/US98/20094 (WO99/15650) filed by Athersys, Inc. Knownas “Random Activation of Gene Expression for Gene Discovery” (RAGE-GD),the process involves the activation of endogenous gene expression orover-expression of a gene by in situ recombination methods. For example,expression of an endogenous gene is activated or increased byintegrating a regulatory sequence into the target cell which is capableof activating expression of the gene by non-homologous or illegitimaterecombination. The target DNA is first subjected to radiation, and agenetic promoter inserted. The promoter eventually locates a break atthe front of a gene, initiating transcription of the gene. This resultsin expression of the desired peptide or polypeptide.

[0213] It will be appreciated that these methods can also be used tocreate comprehensive TSLPR polypeptide expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assays, such as biochemical assays, cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

[0214] Synthesis

[0215] It will be appreciated by those skilled in the art that thenucleic acid and polypeptide molecules described herein may be producedby recombinant and other means.

[0216] Selective Binding Agents

[0217] The term “selective binding agent” refers to a molecule that hasspecificity for one or more TSLPR polypeptides. Suitable selectivebinding agents include, but are not limited to, antibodies andderivatives thereof, polypeptides, and small molecules. Suitableselective binding agents may be prepared using methods known in the art.An exemplary TSLPR polypeptide selective binding agent of the presentinvention is capable of binding a certain portion of the TSLPRpolypeptide thereby inhibiting the binding of the polypeptide to a TSLPRpolypeptide receptor.

[0218] Selective binding agents such as antibodies and antibodyfragments that bind TSLPR polypeptides are within the scope of thepresent invention. The antibodies may be polyclonal includingmonospecific polyclonal; monoclonal (MAbs); recombinant; chimeric;humanized, such as complementarity-determining region (CDR)-grafted;human; single chain; and/or bispecific; as well as fragments; variants;or derivatives thereof. Antibody fragments include those portions of theantibody that bind to an epitope on the TSLPR polypeptide. Examples ofsuch fragments include Fab and F(ab′) fragments generated by enzymaticcleavage of full-length antibodies. Other binding fragments includethose generated by recombinant DNA techniques, such as the expression ofrecombinant plasmids containing nucleic acid sequences encoding antibodyvariable regions.

[0219] Polyclonal antibodies directed toward a TSLPR polypeptidegenerally are produced in animals (e.g., rabbits or mice) by means ofmultiple subcutaneous or intraperitoneal injections of TSLPR polypeptideand an adjuvant. It may be useful to conjugate a TSLPR polypeptide to acarrier protein that is immunogenic in the species to be immunized, suchas keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Also, aggregating agents such as alum areused to enhance the immune response. After immunization, the animals arebled and the serum is assayed for anti-TSLPR antibody titer.

[0220] Monoclonal antibodies directed toward TSLPR polypeptides areproduced using any method that provides for the production of antibodymolecules by continuous cell lines in culture. Examples of suitablemethods for preparing monoclonal antibodies include the hybridomamethods of Kohler et al., 1975, Nature 256:495-97 and the human B-cellhybridoma method (Kozbor, 1984, J. Immunol. 133:3001; Brodeur et al.,Monoclonal Antibody Production Techniques and Applications 51-63 (MarcelDekker, Inc., 1987). Also provided by the invention are hybridoma celllines that produce monoclonal antibodies reactive with TSLPRpolypeptides.

[0221] Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy (H) and/or light (L) chain is identical with or homologousto a corresponding sequence in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is/are identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985,Proc. Natl. Acad. Sci. 81:6851-55.

[0222] In another embodiment, a monoclonal antibody of the invention isa “humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. See U.S. Pat. Nos. 5,585,089 and 5,693,762.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. Humanization can beperformed, for example, using methods described in the art (Jones etal., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27;Verhoeyen et al., 1988, Science 239:1534-36), by substituting at least aportion of a rodent complementarity-determining region for thecorresponding regions of a human antibody.

[0223] Also encompassed by the invention are human antibodies that bindTSLPR polypeptides. Using transgenic animals (e.g., mice) that arecapable of producing a repertoire of human antibodies in the absence ofendogenous immunoglobulin production such antibodies are produced byimmunization with a TSLPR polypeptide antigen (i.e., having at least 6contiguous amino acids), optionally conjugated to a carrier. See, e.g.,Jakobovits et al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovitset al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year inImmuno. 7:33. In one method, such transgenic animals are produced byincapacitating the endogenous loci encoding the heavy and lightimmunoglobulin chains therein, and inserting loci encoding human heavyand light chain proteins into the genome thereof. Partially modifiedanimals, that is those having less than the full complement ofmodifications, are then cross-bred to obtain an animal having all of thedesired immune system modifications. When administered an immunogen,these transgenic animals produce antibodies with human (rather than,e.g., murine) amino acid sequences, including variable regions which areimmunospecific for these antigens. See PCT App. Nos. PCT/US96/05928 andPCT/US93/06926. Additional methods are described in U.S. Pat. No.5,545,807, PCT App. Nos.PCT/US91/245 and PCT/GB89/01207, and in EuropeanPatent Nos. 546073B1 and 546073A1. Human antibodies can also be producedby the expression of recombinant DNA in TSLPR polyhost cells or byexpression in hybridoma cells as described herein.

[0224] In an alternative embodiment, human antibodies can also beproduced from phage-display libraries (Hoogenboom et al., 1991, J. Mol.Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581). Theseprocesses mimic immune selection through the display of antibodyrepertoires on the surface of filamentous bacteriophage, and subsequentselection of phage by their binding to an antigen of choice. One suchtechnique is described in PCT App. No. PCT/US98/17364, which describesthe isolation of high affinity and functional agonistic antibodies forMPL- and msk-receptors using such an approach.

[0225] Chimeric, CDR grafted, and humanized antibodies are typicallyproduced by recombinant methods. Nucleic acids encoding the antibodiesare introduced into host cells and expressed using materials andprocedures described herein. In a preferred embodiment, the antibodiesare produced in mammalian host cells, such as CHO cells. Monoclonal(e.g., human) antibodies may be produced by the expression ofrecombinant DNA in host cells or by expression in hybridoma cells asdescribed herein.

[0226] The anti-TSLPR antibodies of the invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (Sola,Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC Press, Inc.,1987)) for the detection and quantitation of TSLPR polypeptides. Theantibodies will bind TSLPR polypeptides with an affinity that isappropriate for the assay method being employed.

[0227] For diagnostic applications, in certain embodiments, anti-TSLPRantibodies may be labeled with a detectable moiety. The detectablemoiety can be any one that is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, 14C, ³²P, ³⁵S, 125I, ⁹⁹Tc, ¹¹¹In, or⁶⁷Ga; a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase, or horseradish peroxidase (Bayer, et al.,1990, Meth. Enz. 184:138-63).

[0228] Competitive binding assays rely on the ability of a labeledstandard (e.g., a TSLPR polypeptide, or an immunologically reactiveportion thereof) to compete with the test sample analyte (an TSLPRpolypeptide) for binding with a limited amount of anti-TSLPR antibody.The amount of a TSLPR polypeptide in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies typically are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

[0229] Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is typically bound by a first antibody which isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three-part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme4inked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme.

[0230] The selective binding agents, including anti-TSLPR antibodies,are also useful for in vivo imaging. An antibody labeled with adetectable moiety may be administered to an animal, preferably into thebloodstream, and the presence and location of the labeled antibody inthe host assayed. The antibody may be labeled with any moiety that isdetectable in an animal, whether by nuclear magnetic resonance,radiology, or other detection means known in the art.

[0231] Selective binding agents of the invention, including antibodies,may be used as therapeutics. These therapeutic agents are generallyagonists or antagonists, in that they either enhance or reduce,respectively, at least one of the biological activities of a TSLPRpolypeptide. In one embodiment, antagonist antibodies of the inventionare antibodies or binding fragments thereof which are capable ofspecifically binding to a TSLPR polypeptide and which are capable ofinhibiting or eliminating the functional activity of a TSLPR polypeptidein vivo or in vitro. In preferred embodiments, the selective bindingagent, e.g., an antagonist antibody, will inhibit the functionalactivity of a TSLPR polypeptide by at least about 50%, and preferably byat least about 80%. In another embodiment, the selective binding agentmay be an anti-TSLPR polypeptide antibody that is capable of interactingwith a TSLPR polypeptide binding partner (a ligand or receptor) therebyinhibiting or eliminating TSLPR polypeptide activity in vitro or invivo. Selective binding agents, including agonist and antagonistanti-TSLPR polypeptide antibodies, are identified by screening assaysthat are well known in the art.

[0232] The invention also relates to a kit comprising TSLPR selectivebinding agents (such as antibodies) and other reagents useful fordetecting TSLPR polypeptide levels in biological samples. Such reagentsmay include a detectable label, blocking serum, positive and negativecontrol samples, and detection reagents.

[0233] Microarrays

[0234] It will be appreciated that DNA microarray technology can beutilized in accordance with the present invention. DNA microarrays areminiature, high-density arrays of nucleic acids positioned on a solidsupport, such as glass. Each cell or element within the array containsnumerous copies of a single nucleic acid species that acts as a targetfor hybridization with a complementary nucleic acid sequence (e.g.,mRNA). In expression profiling using DNA microarray technology, mRNA isfirst extracted from a cell or tissue sample and then convertedenzymatically to fluorescently labeled cDNA. This material is hybridizedto the microarray and unbound cDNA is removed by washing. The expressionof discrete genes represented on the array is then visualized byquantitating the amount of labeled EDNA that is specifically bound toeach target nucleic acid molecule. In this way, the expression ofthousands of genes can be quantitated in a high throughput, parallelmanner from a single sample of biological material.

[0235] This high throughput expression profiling has a broad range ofapplications with respect to the TSLPR molecules of the invention,including, but not limited to: the identification and validation ofTSLPR disease-related genes as targets for therapeutics; moleculartoxicology of related TSLPR molecules and inhibitors thereof,stratification of populations and generation of surrogate markers forclinical trials; and enhancing related TSLPR polypeptide small moleculedrug discovery by aiding in the identification of selective compounds inhigh throughput screens.

[0236] Chemical Derivatives

[0237] Chemically modified derivatives of TSLPR polypeptides may beprepared by one skilled in the art, given the disclosures describedherein. TSLPR polypeptide derivatives are modified in a manner that isdifferent—either in the type or location of the molecules naturallyattached to the polypeptide. Derivatives may include molecules formed bythe deletion of one or more naturally-attached chemical groups. Thepolypeptide comprising the amino acid sequence of any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8, or other TSLPR polypeptide, may bemodified by the covalent attachment of one or more polymers. Forexample, the polymer selected is typically water-soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. Included within thescope of suitable polymers is a mixture of polymers. Preferably, fortherapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

[0238] The polymers each may be of any molecular weight and may bebranched or unbranched. The polymers each typically have an averagemolecular weight of between about 2 kDa to about 100 kDa (the term“about” indicating that in preparations of a water-soluble polymer, somemolecules will weigh more, some less, than the stated molecular weight).The average molecular weight of each polymer is preferably between about5 kDa and about 50 kDa, more preferably between about 12 kDa and about40 kDa and most preferably between about 20 kDa and about 35 kDa.

[0239] Suitable water-soluble polymers or mixtures thereof include, butare not limited to, N-linked or O-linked carbohydrates, sugars,phosphates, polyethylene glycol (PEG) (including the forms of PEG thathave been used to derivatize proteins, including mono-(C₁-C₁₀), alkoxy-,or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol,dextran (such as low molecular weight dextran of, for example, about 6kD), cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached TSLPR polypeptide multimers.

[0240] In general, chemical derivatization may be performed under anysuitable condition used to react a protein with an activated polymermolecule. Methods for preparing chemical derivatives of polypeptideswill generally comprise the steps of: (a) reacting the polypeptide withthe activated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide comprising the amino acid sequence of any of SEQ ID NO: 2,SEQ ID NO: 5, or SEQ ID NO: 8, or other TSLPR polypeptide, becomesattached to one or more polymer molecules, and (b) obtaining thereaction products. The optimal reaction conditions will be determinedbased on known parameters and the desired result. For example, thelarger the ratio of polymer molecules to protein, the greater thepercentage of attached polymer molecule. In one embodiment, the TSLPRpolypeptide derivative may have a single polymer molecule moiety at theamino-terminus. See, e.g., U.S. Pat. No. 5,234,784.

[0241] The pegylation of a polypeptide may be specifically carried outusing any of the pegylation reactions known in the art. Such reactionsare described, for example, in the following references: Francis et al.,1992, Focus on Growth Factors 3:4-10;

[0242] European Patent Nos. 0154316 and 0401384; and U.S. Pat. No.4,179,337. For example, pegylation may be carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer) as describedherein. For the acylation reactions, a selected polymer should have asingle reactive ester group. For reductive alkylation, a selectedpolymer should have a single reactive aldehyde group. A reactivealdehyde is, for example, polyethylene glycol propionaldehyde, which iswater stable, or mono C₁-C₁₀ alkoxy or aryloxy derivatives thereof (seeU.S. Pat. No. 5,252,714).

[0243] In another embodiment, TSLPR polypeptides may be chemicallycoupled to biotin. The biotin/TSLPR polypeptide molecules are thenallowed to bind to avidin, resulting in tetravalent avidin/biotin/TSLPRpolypeptide molecules. TSLPR polypeptides may also be covalently coupledto dinitrophenol (DNP) or trinitrophenol (TNP) and the resultingconjugates precipitated with anti-DNP or anti-TNP-IgM to form decamericconjugates with a valency of 10.

[0244] Generally, conditions that may be alleviated or modulated by theadministration of the present TSLPR polypeptide derivatives includethose described herein for TSLPR polypeptides. However, the TSLPRpolypeptide derivatives disclosed herein may have additional activities,enhanced or reduced biological activity, or other characteristics, suchas increased or decreased half-life, as compared to the non-derivatizedmolecules.

[0245] Genetically Engineered Non-Human Animals

[0246] Additionally included within the scope of the present inventionare non-human animals such as mice, rats, or other rodents; rabbits,goats, sheep, or other farm animals, in which the genes encoding nativeTSLPR polypeptide have been TSLPR polydisrupted (i.e., “knocked out”)such that the level of expression of TSLPR polypeptide is significantlydecreased or completely abolished. Such animals may be prepared usingtechniques and methods such as those described in U.S. Pat. No.5,557,032.

[0247] The present invention further includes non-human animals such asmice, rats, or other rodents; rabbits, goats, sheep, or other farmanimals, in which either the native form of a TSLPR gene for that animalor a heterologous TSLPR gene is over-expressed by the animal, therebycreating a “transgenic” animal. Such transgenic animals may be preparedusing well known methods such as those described in U.S. Pat. No5,489,743 and PCT Pub. No. WO 94/28122.

[0248] The present invention further includes non-human animals in whichthe promoter for one or more of the TSLPR polypeptides of the presentinvention is either activated or inactivated (e.g., by using homologousrecombination methods) to alter the level of expression of one or moreof the native TSLPR polypeptides.

[0249] These non-human animals may be used for drug candidate screening.In such screening, the impact of a drug candidate on the animal may bemeasured. For example, drug candidates may decrease or increase theexpression of the TSLPR gene. In certain embodiments, the amount ofTSLPR polypeptide that is produced may be measured after the exposure ofthe animal to the drug candidate. Additionally, in certain embodiments,one may detect the actual impact of the drug candidate on the animal.For example, over-expression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

[0250] Assaying for Other Modulators of TSLPR Polypeptide Activity

[0251] In some situations, it may be desirable to identify moleculesthat are modulators, i.e., agonists or antagonists, of the activity ofTSLPR polypeptide. Natural or synthetic molecules that modulate TSLPRpolypeptide may be identified using one or more screening assays, suchas those described herein. Such molecules may be administered either inan ex vivo manner or in an in vivo manner by injection, or by oraldelivery, implantation device, or the like.

[0252] “Test molecule” refers to a molecule that is under evaluation forthe ability to modulate (i.e., increase or decrease) the activity of aTSLPR polypeptide. Most commonly, a test molecule will interact directlywith a TSLPR polypeptide. However, it is also contemplated that a testmolecule may also modulate TSLPR polypeptide activity indirectly, suchas by affecting TSLPR gene expression, or by binding to a TSLPRpolypeptide binding partner (e.g., receptor or ligand). In oneembodiment, a test molecule will bind to a TSLPR polypeptide with anaffinity constant of at least about 10⁻⁶ M, preferably about 10⁻⁸ M,more preferably about 10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

[0253] Methods for identifying compounds that interact with TSLPRpolypeptides are encompassed by the present invention. In certainembodiments, a TSLPR polypeptide is incubated with a test molecule underconditions that permit the interaction of the test molecule with a TSLPRpolypeptide, and the extent of the interaction is measured. The testmolecule can be screened in a substantially purified form or in a crudemixture.

[0254] In certain embodiments, a TSLPR polypeptide agonist or antagonistmay be a protein, peptide, carbohydrate, lipid, or small molecularweight molecule that interacts with TSLPR polypeptide to regulate itsactivity. Molecules which regulate TSLPR polypeptide expression includenucleic acids which are complementary to nucleic acids encoding a TSLPRpolypeptide, or are complementary to nucleic acids sequences whichdirect or control the expression of TSLPR polypeptide, and which act asanti-sense regulators of expression.

[0255] Once a test molecule has been identified as interacting with aTSLPR polypeptide, the molecule may be further evaluated for its abilityto increase or decrease TSLPR polypeptide activity. The measurement ofthe interaction of a test molecule with TSLPR polypeptide may be carriedout in several formats, including cell-based binding assays, membranebinding assays, solution-phase assays, and immunoassays. In general, atest molecule is incubated with a TSLPR polypeptide for a specifiedperiod of time, and TSLPR polypeptide activity is determined by one ormore assays for measuring biological activity.

[0256] The interaction of test molecules with TSLPR polypeptides mayalso be assayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of TSLPR polypeptidescontaining epitope tags as described herein may be used in solution andimmunoassays.

[0257] In the event that TSLPR polypeptides display biological activitythrough an interaction with a binding partner (e.g., a receptor or aligand), a variety of in vitro assays may be used to measure the bindingof a TSLPR polypeptide to the corresponding binding partner (such as aselective binding agent, receptor, or ligand). These assays may be usedto screen test molecules for their ability to increase or decrease therate and/or the extent of binding of a TSLPR polypeptide to its bindingpartner. In one assay, a TSLPR polypeptide is immobilized in the wellsof a microtiter plate. Radiolabeled TSLPR polypeptide binding partner(for example, iodinated TSLPR polypeptide binding partner) and a testmolecule can then be added either one at a time (in either order) orsimultaneously to the wells. After incubation, the wells can be washedand counted for radioactivity, using a scintillation counter, todetermine the extent to which the binding partner bound to the TSLPRpolypeptide. Typically, a molecule will be tested over a range ofconcentrations, and a series of control wells lacking one or moreelements of the test assays can be used for accuracy in the evaluationof the results. An alternative to this method involves reversing the“positions” of the proteins, i.e., immobilizing TSLPR polypeptidebinding partner to the microtiter plate wells, incubating with the testmolecule and radiolabeled TSLPR polypeptide, and determining the extentof TSLPR polypeptide binding. See, e.g., Current Protocols in MolecularBiology, chap. 18 (Ausubel et al., eds., Green Publishers Inc. and Wileyand Sons 1995).

[0258] As an alternative to radiolabeling, a TSLPR polypeptide or itsbinding partner may be conjugated to biotin, and the presence ofbiotinylated protein can then be detected using streptavidin linked toan enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase(AP), which can be detected colorometrically, or by fluorescent taggingof streptavidin. An antibody directed to a TSLPR polypeptide or to aTSLPR polypeptide binding partner, and which is conjugated to biotin,may also be used for purposes of detection following incubation of thecomplex with enzyme-linked streptavidin linked to AP or HRP.

[0259] A TSLPR polypeptide or a TSLPR polypeptide binding partner canalso be immobilized by attachment to agarose beads, acrylic beads, orother types of such inert solid phase substrates. The substrate-proteincomplex can be placed in a solution containing the complementary proteinand the test compound. After incubation, the beads can be precipitatedby centrifugation, and the amount of binding between a TSLPR polypeptideand its binding partner can be assessed using the methods describedherein. Alternatively, the substrate-protein complex can be immobilizedin a column with the test molecule and complementary protein passingthrough the column. The formation of a complex between a TSLPRpolypeptide and its binding partner can then be assessed using any ofthe techniques described herein (e.g., radiolabelling or antibodybinding).

[0260] Another in vitro assay that is useful for identifying a testmolecule that increases or decreases the formation of a complex betweena TSLPR polypeptide binding protein and a TSLPR polypeptide bindingpartner is a surface plasmon resonance detector system such as theBIAcore assay system (Pharmacia, Piscataway, N.J.). The BIAcore systemis utilized as specified by the manufacturer. This assay essentiallyinvolves the covalent binding of either TSLPR polypeptide or a TSLPRpolypeptide binding partner to a dextran-coated sensor chip that islocated in a detector. The test compound and the other complementaryprotein can then be injected, either simultaneously or sequentially,into the chamber containing the sensor chip. The amount of complementaryprotein that binds can be assessed based on the change in molecular massthat is physically associated with the dextran-coated side of the sensorchip, with the change in molecular mass being measured by the detectorsystem.

[0261] In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between a TSLPR polypeptide and a TSLPRpolypeptide binding partner. In these cases, the assays set forth hereincan be readily modified by adding such additional test compound(s)either simultaneously with, or subsequent to, the first test compound.The remainder of the steps in the assay are as set forth herein.

[0262] In vitro assays such as those described herein may be usedadvantageously to screen large numbers of compounds for an effect on theformation of a complex between a TSLPR polypeptide and TSLPR polypeptidebinding partner. The assays may be automated to screen compoundsgenerated in phage display, synthetic peptide, and chemical synthesislibraries.

[0263] Compounds which increase or decrease the formation of a complexbetween a TSLPR polypeptide and a TSLPR polypeptide binding partner mayalso be screened in cell culture using cells and cell lines expressingeither TSLPR polypeptide or TSLPR polypeptide binding partner. Cells andcell lines may be obtained from any mammal, but preferably will be fromhuman or other primate, canine, or rodent sources. The binding of aTSLPR polypeptide to cells expressing TSLPR polypeptide binding partnerat the surface is evaluated in the presence or absence of testmolecules, and the extent of binding may be determined by, for example,flow cytometry using a biotinylated antibody to a TSLPR polypeptidebinding partner. Cell culture assays can be used advantageously tofurther evaluate compounds that score positive in protein binding assaysdescribed herein.

[0264] Cell cultures can also be used to screen the impact of a drugcandidate. For example, drug candidates may decrease or increase theexpression of the TSLPR gene. In certain embodiments, the amount ofTSLPR polypeptide or a TSLPR polypeptide fragment that is produced maybe measured after exposure of the cell culture to the drug candidate. Incertain embodiments, one may detect the actual impact of the drugcandidate on the cell culture. For example, the over-expression of aparticular gene may have a particular impact on the cell culture. Insuch cases, one may test a drug candidate's ability to increase ordecrease the expression of the gene or its ability to prevent or inhibita particular impact on the cell culture. In other examples, theproduction of a particular metabolic product such as a fragment of apolypeptide, may result in, or be associated with, a disease orpathological condition.

[0265] In such cases, one may test a drug candidate's ability todecrease the production of such a metabolic product in a cell culture.

[0266] Internalizing Proteins

[0267] The tat protein sequence (from HIV) can be used to internalizeproteins into a cell. See, e.g., Falwell et al., 1994, Proc. Natl. Acad.Sci. U.S.A. 91:664-68. For example, an 11 amino acid sequence(Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 13) of the HIV tat protein (termedthe “protein transduction domain,” or TAT PDT) has been described asmediating delivery across the cytoplasmic membrane and the nuclearmembrane of a cell. See Schwarze et al., 1999, Science 285:1569-72; andNagahara et al., 1998, Nat. Med. 4:1449-52. In these procedures,FITC-constructs (FITC-labeled G-G-G-G-Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO:14), which penetrate tissues following intraperitoneal administration,are prepared, and the binding of such constructs to cells is detected byfluorescence-activated cell sorting (FACS) analysis. Cells treated witha tat-β-gal fusion protein will demonstrate β-gal activity. Followinginjection, expression of such a construct can be detected in a number oftissues, including liver, kidney, lung, heart, and brain tissue. It isbelieved that such constructs undergo some degree of unfolding in orderto enter the cell, and as such, may require a refolding following entryinto the cell.

[0268] It will thus be appreciated that the tat protein sequence may beused to internalize a desired polypeptide into a cell. For example,using the tat protein sequence, a TSLPR antagonist (such as ananti-TSLPR selective binding agent, small molecule, soluble receptor, orantisense oligonucleotide) can be administered intracellularly toinhibit the activity of a TSLPR molecule. As used herein, the term“TSLPR molecule” refers to both TSLPR nucleic acid molecules and TSLPRpolypeptides as defined herein. Where desired, the TSLPR protein itselfmay also be internally administered to a cell using these procedures.See also, Straus, 1999, Science 285:1466-67.

[0269] Cell Source Identification Using TSLPR Polypeptide

[0270] In accordance with certain embodiments of the invention, it maybe useful to be able to determine the source of a certain cell typeassociated with a TSLPR polypeptide. For example, it may be useful todetermine the origin of a disease or pathological condition as an aid inselecting an appropriate therapy. In certain embodiments, nucleic acidsencoding a TSLPR polypeptide can be used as a probe to identify cellsdescribed herein by screening the nucleic acids of the cells with such aprobe. In other embodiments, one may use anti-TSLPR polypeptideantibodies to test for the presence of TSLPR polypeptide in cells, andthus, determine if such cells are of the types described herein.

[0271] TSLPR Polypeptide Compositions and Administration

[0272] Therapeutic compositions are within the scope of the presentinvention. Such TSLPR polypeptide pharmaceutical compositions maycomprise a therapeutically effective amount of a TSLPR polypeptide or aTSLPR nucleic acid molecule in admixture with a pharmaceutically orphysiologically acceptable formulation agent selected for suitabilitywith the mode of administration. Pharmaceutical compositions maycomprise a therapeutically effective amount of one or more TSLPRpolypeptide selective binding agents in admixture with apharmaceutically or physiologically acceptable formulation agentselected for suitability with the mode of administration.

[0273] Acceptable formulation materials preferably are nontoxic torecipients at the dosages and concentrations employed.

[0274] The pharmaceutical composition may contain formulation materialsfor modifying, maintaining, or preserving, for example, the pH,osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption, or penetration ofthe composition. Suitable formulation materials include, but are notlimited to, amino acids (such as glycine, glutamine, asparagine,arginine, or lysine), antimicrobials, antioxidants (such as ascorbicacid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such asborate, bicarbonate, Tris-HCl, citrates, phosphates, or other organicacids), bulking agents (such as mannitol or glycine), chelating agents(such as ethylenediamine tetraacetic acid (EDTA)), complexing agents(such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin,gelatin, or immunoglobulins),coloring, flavoring and diluting agents, emulsifying agents, hydrophilicpolymers (such as polyvinylpyrrolidone), low molecular weightpolypeptides, salt-forming counterions (such as sodium), preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such asglycerin, propylene glycol, or polyethylene glycol), sugar alcohols(such as mannitol or sorbitol), suspending agents, surfactants orwetting agents (such as pluronics; PEG; sorbitan esters; polysorbatessuch as polysorbate 20 or polysorbate 80; triton; tromethamine;lecithin; cholesterol or tyloxapal), stability enhancing agents (such assucrose or sorbitol), tonicity enhancing agents (such as alkali metalhalides -preferably sodium or potassium chloride-or mannitol sorbitol),delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.See Remington's Pharmaceutical Sciences (18th Ed., A. R. Gennaro, ed.,Mack Publishing Company 1990.

[0275] The optimal pharmaceutical composition will be determined by askilled artisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See, e.g.,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the TSLPR molecule.

[0276] The primary vehicle or carrier in a pharmaceutical compositionmay be either aqueous or non-aqueous in nature. For example, a suitablevehicle or carrier for injection may be water, physiological salinesolution, or artificial cerebrospinal fluid, possibly supplemented withother materials common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin are furtherexemplary vehicles. Other exemplary pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,which may further include sorbitol or a suitable substitute. In oneembodiment of the present invention, TSLPR polypeptide compositions maybe prepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the TSLPR polypeptide product may beformulated as a lyophilizate using appropriate excipients such assucrose.

[0277] The TSLPR polypeptide pharmaceutical compositions can be selectedfor parenteral delivery. Alternatively, the compositions may be selectedfor inhalation or for delivery through the digestive tract, such asorally. The preparation of such pharmaceutically acceptable compositionsis within the skill of the art.

[0278] The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

[0279] When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable, aqueous solution comprising thedesired TSLPR molecule in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which a TSLPR molecule is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads, or liposomes,that provides for the controlled or sustained release of the productwhich may then be delivered via a depot injection. Hyaluronic acid mayalso be used, and this may have the effect of promoting sustainedduration in the circulation. Other suitable means for the introductionof the desired molecule include implantable drug delivery devices.

[0280] In one embodiment, a pharmaceutical composition may be formulatedfor inhalation. For example, TSLPR polypeptide may be formulated as adry powder for inhalation. TSLPR polypeptide or nucleic acid moleculeinhalation solutions may also be formulated with a propellant foraerosol delivery. In yet another embodiment, solutions may be nebulized.Pulmonary administration is further described in PCT Pub. No. WO94/20069, which describes the pulmonary delivery of chemically modifiedproteins.

[0281] It is also contemplated that certain formulations may beadministered orally. In one embodiment of the present invention, TSLPRpolypeptides that are administered in this fashion can be formulatedwith or without those carriers customarily used in the compounding ofsolid dosage forms such as tablets and capsules. For example, a capsulemay be designed to release the active portion of the formulation at thepoint in the gastrointestinal tract when bioavailability is maximizedand pre-systemic degradation is minimized. Additional agents can beincluded to facilitate absorption of the TSLPR polypeptide. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed.

[0282] Another pharmaceutical composition may involve an effectivequantity of TSLPR polypeptides in a mixture with non-toxic excipientsthat are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or another appropriate vehicle, solutions canbe prepared in unit-dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

[0283] Additional TSLPR polypeptide pharmaceutical compositions will beevident to those skilled in the art, including formulations involvingTSLPR polypeptides in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, e.g., PCT/US93/00829, which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions.

[0284] Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 andEuropean Pat. No. 058481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-56),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(-)-3-hydroxybutyric acid (European Pat. No. 133988).Sustained-release compositions may also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Eppsteinet al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; and European PatentNos. 036676, 088046, and 143949.

[0285] The TSLPR pharmaceutical composition to be used for in vivoadministration typically must be sterile. This may be accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using this method may be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration may be stored in lyophilizedform or in a solution. In addition, parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

[0286] Once the pharmaceutical composition has been formulated, it maybe stored in sterile vials as a solution, suspension, gel, emulsion,solid, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

[0287] In a specific embodiment, the present invention is directed tokits for producing a single-dose administration unit. The kits may eachcontain both a first container having a dried protein and a secondcontainer having an aqueous formulation. Also included within the scopeof this invention are kits containing single and multi-chamberedpre-filled syringes (e.g., liquid syringes and lyosyringes).

[0288] The effective amount of a TSLPR pharmaceutical composition to beemployed therapeutically will depend, for example, upon the therapeuticcontext and objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the TSLPRmolecule is being used, the route of administration, and the size (bodyweight, body surface, or organ size) and condition (the age and generalhealth) of the patient. Accordingly, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 μg/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 ∥g/kg up to about 100 mg/kg;or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg.

[0289] The frequency of dosing will depend upon the pharmacokineticparameters of the TSLPR molecule in the formulation being used.Typically, a clinician will administer the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

[0290] The route of administration of the pharmaceutical composition isin accord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial,intraportal, or intralesional routes; by sustained release systems; orby implantation devices. Where desired, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

[0291] Alternatively or additionally, the composition may beadministered locally via implantation of a membrane, sponge, or otherappropriate material onto which the desired molecule has been absorbedor encapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

[0292] In some cases, it may be desirable to use TSLPR polypeptidepharmaceutical compositions in an ex vivo manner. In such instances,cells, tissues, or organs that have been removed from the patient areexposed to TSLPR polypeptide pharmaceutical compositions after which thecells, tissues, or organs are subsequently implanted back into thepatient.

[0293] In other cases, a TSLPR polypeptide can be delivered byimplanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete the TSLPRpolypeptide. Such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic. Optionally, the cells may beimmortalized. In order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues.

[0294] The encapsulation materials are typically biocompatible,semi-permeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

[0295] As discussed herein, it may be desirable to treat isolated cellpopulations (such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like) with one or more TSLPRpolypeptides. This can be accomplished by exposing the isolated cells tothe polypeptide directly, where it is in a form that is permeable to thecell membrane.

[0296] Additional embodiments of the present invention relate to cellsand methods (e.g., homologous recombination and/or other recombinantproduction methods) for both the in vitro production of therapeuticpolypeptides and for the production and delivery of therapeuticpolypeptides by gene therapy or cell therapy. Homologous and otherrecombination methods may be used to modify a cell that contains anormally transcriptionally-silent TSLPR gene, or an under-expressedgene, and thereby produce a cell which expresses therapeuticallyefficacious amounts of TSLPR polypeptides.

[0297] Homologous recombination is a technique originally developed fortargeting genes to induce or correct mutations in transcriptionallyactive genes. Kucherlapati, 1989, Prog. in Nucl. Acid Res. & Mol. Biol.36:301. The basic technique was developed as a method for introducingspecific mutations into specific regions of the mammalian genome (Thomaset al., 1986, Cell 44:419-28; Thomas and Capecchi, 1987, Cell 51:503-12;Doetschman et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8583-87) or tocorrect specific mutations within defective genes (Doetschman et al.,1987, Nature 330:576-78). Exemplary homologous recombination techniquesare described in U.S. Pat. No. 5,272,071; European Patent Nos. 9193051and 505500; PCT/US90/07642, and PCT Pub No. WO 91/09955).

[0298] Through homologous recombination, the DNA sequence to be insertedinto the genome can be directed to a specific region of the gene ofinterest by attaching it to targeting DNA. The targeting DNA is anucleotide sequence that is complementary (homologous) to a region ofthe genomic DNA. Small pieces of targeting DNA that are complementary toa specific region of the genome are put in contact with the parentalstrand during the DNA replication process. It is a general property ofDNA that has been inserted into a cell to hybridize, and therefore,recombine with other pieces of endogenous DNA through shared homologousregions. If this complementary strand is attached to an oligonucleotidethat contains a mutation or a different sequence or an additionalnucleotide, it too is incorporated into the newly synthesized strand asa result of the recombination. As a result of the proofreading function,it is possible for the new sequence of DNA to serve as the template.Thus, the transferred DNA is incorporated into the genome.

[0299] Attached to these pieces of targeting DNA are regions of DNA thatmay interact with or control the expression of a TSLPR polypeptide,e.g., flanking sequences. For example, a promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredTSLPR polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of the desiredTSLPR polypeptide may be achieved not by transfection of DNA thatencodes the TSLPR gene itself, but rather by the use of targeting DNA(containing regions of homology with the endogenous gene of interest)coupled with DNA regulatory segments that provide the endogenous genesequence with recognizable signals for transcription of a TSLPR gene.

[0300] In an exemplary method, the expression of a desired targeted genein a cell (i.e., a desired endogenous cellular gene) is altered viahomologous recombination into the cellular genome at a preselected site,by the introduction of DNA which includes at least a regulatorysequence, an exon, and a splice donor site. These components areintroduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon, and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

[0301] Altered gene expression, as described herein, encompassesactivating (or causing to be expressed) a gene which is normally silent(unexpressed) in the cell as obtained, as well as increasing theexpression of a gene which is not expressed at physiologicallysignificant levels in the cell as obtained. The embodiments furtherencompass changing the pattern of regulation or induction such that itis different from the pattern of regulation or induction that occurs inthe cell as obtained, and reducing (including eliminating) theexpression of a gene which is expressed in the cell as obtained.

[0302] One method by which homologous recombination can be used toincrease, or cause, TSLPR polypeptide production from a cell'sendogenous TSLPR gene involves first using homologous recombination toplace a recombination sequence from a site-specific recombination system(e.g., Cre/loxP, FLP/FRT) (Sauer, 1994, Curr. Opin. Biotechnol.,5:521-27; Sauer, 1993,Methods Enzymol., 225:890-900) upstream of (i.e.,5′ to) the cell's endogenous genomic TSLPR polypeptide coding region. Aplasmid containing a recombination site homologous to the site that wasplaced just upstream of the genomic TSLPR polypeptide coding region isintroduced into the modified cell line along with the appropriaterecombinase enzyme. This recombinase causes the plasmid to integrate,via the plasmid's recombination site, into the recombination sitelocated just upstream of the genomic TSLPR polypeptide coding region inthe cell line (Baubonis and Sauer, 1993,Nucleic Acids Res. 21:2025-29;O'Gorman et al., 1991, Science 251:1351-55). Any flanking sequencesknown to increase transcription (e.g., enhancer/promoter, intron,translational enhancer), if properly positioned in this plasmid, wouldintegrate in such a manner as to create a new or modifiedtranscriptional unit resulting in de novo or increased TSLPR polypeptideproduction from the cell's endogenous TSLPR gene.

[0303] A further method to use the cell line in which the site specificrecombination sequence had been placed just upstream of the cell'sendogenous genomic TSLPR polypeptide coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, and translocation) (Sauer,1994, Curr. Opin. Biotechnol., 5:521-27; Sauer, 1993,Methods Enzymol.,225:890-900) that would create a new or modified transcriptional unitresulting in de novo or increased TSLPR polypeptide production from thecell's endogenous TSLPR gene.

[0304] An additional approach for increasing, or causing, the expressionof TSLPR polypeptide from a cell's endogenous TSLPR gene involvesincreasing, or causing, the expression of a gene or genes (e.g.,transcription factors) and/or decreasing the expression of a gene orgenes (e.g., transcriptional repressors) in a manner which results in denovo or increased TSLPR polypeptide production from the cell'sendogenous TSLPR gene. This method includes the introduction of anon-naturally occurring polypeptide (e.g., a polypeptide comprising asite specific DNA binding domain fused to a transcriptional factordomain) into the cell such that de novo or increased TSLPR polypeptideproduction from the cell's endogenous TSLPR gene results.

[0305] The present invention further relates to DNA constructs useful inthe method of altering expression of a target gene. In certainembodiments, the exemplary DNA constructs comprise: (a) one or moretargeting sequences, (b) a regulatory sequence, (c) an exon, and (d) anunpaired splice-donor site. The targeting sequence in the DNA constructdirects the integration of elements (a)-(d) into a target gene in a cellsuch that the elements (b)-(d) are operatively linked to sequences ofthe endogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequences, (b) a regulatorysequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

[0306] If the sequence of a particular gene is known, such as thenucleic acid sequence of TSLPR polypeptide presented herein, a piece ofDNA that is complementary to a selected region of the gene can besynthesized or otherwise obtained, such as by appropriate restriction ofthe native DNA at specific recognition sites bounding the region ofinterest. This piece serves as a targeting sequence upon insertion intothe cell and will hybridize to its homologous region within the genome.If this hybridization occurs during DNA replication, this piece of DNA,and any additional sequence attached thereto, will act as an Okazakifragment and will be incorporated into the newly synthesized daughterstrand of DNA. The present invention, therefore, includes nucleotidesencoding a TSLPR polypeptide, which nucleotides may be used as targetingsequences.

[0307] TSLPR polypeptide cell therapy, e.g., the implantation of cellsproducing TSLPR polypeptides, is also contemplated. This embodimentinvolves implanting cells capable of synthesizing and secreting abiologically active form of TSLPR polypeptide. Such TSLPRpolypeptide-producing cells can be cells that are natural producers ofTSLPR polypeptides or may be recombinant cells whose ability to produceTSLPR polypeptides has been augmented by transformation with a geneencoding the desired TSLPR polypeptide or with a gene augmenting theexpression of TSLPR polypeptide. Such a modification may be accomplishedby means of a vector suitable for delivering the gene as well aspromoting its expression and secretion. In order to minimize a potentialimmunological reaction in patients being administered a TSLPRpolypeptide, as may occur with the administration of a polypeptide of aforeign species, it is preferred that the natural cells producing TSLPRpolypeptide be of human origin and produce human TSLPR polypeptide.Likewise, it is preferred that the recombinant cells producing TSLPRpolypeptide be transformed with an expression vector containing a geneencoding a human TSLPR polypeptide.

[0308] Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow the release of TSLPR polypeptide, but that preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue.

[0309] Alternatively, the patient's own cells, transformed to produceTSLPR polypeptides ex vivo, may be implanted directly into the patientwithout such encapsulation.

[0310] Techniques for the encapsulation of living cells are known in theart, and the preparation of the encapsulated cells and theirimplantation in patients may be routinely accomplished. For example,Baetge et al. (PCT Pub. No. WO 95/05452 and PCT/US94/09299) describemembrane capsules containing genetically engineered cells for theeffective delivery of biologically active molecules. The capsules arebiocompatible and are easily retrievable. The capsules encapsulate cellstransfected with recombinant DNA molecules comprising DNA sequencescoding for biologically active molecules operatively linked to promotersthat are not subject to down-regulation in vivo upon implantation into amammalian host. The devices provide for the delivery of the moleculesfrom living cells to specific sites within a recipient. In addition, seeU.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627. A system forencapsulating living cells is described in PCT Pub. No. WO 91/10425(Aebischeret al.). See also, PCT Pub. No. WO 91/10470 (Aebischeret al.);Winn et al., 1991, Exper. Neurol. 113:322-29; Aebischer et al., 1991,Exper. Neurol. 111:269-75; and Tresco et al., 1992, ASAIO 38:17-23.

[0311] In vivo and in vitro gene therapy delivery of TSLPR polypeptidesis also envisioned. One example of a gene therapy technique is to usethe TSLPR gene (either genomic DNA, cDNA, and/or synthetic DNA) encodinga TSLPR polypeptide which may be operably linked to a constitutive orinducible promoter to form a “gene therapy DNA construct.” The promotermay be homologous or heterologous to the endogenous TSLPR gene, providedthat it is active in the cell or tissue type into which the constructwill be inserted. Other components of the gene therapy DNA construct mayoptionally include DNA molecules designed for site-specific integration(e.g., endogenous sequences useful for homologous recombination),tissue-specific promoters, enhancers or silencers, DNA molecules capableof providing a selective advantage over the parent cell, DNA moleculesuseful as labels to identify transformed cells, negative selectionsystems, cell specific binding agents (as, for example, for celltargeting), cell-specific internalization factors, transcription factorsenhancing expression from a vector, and factors enabling vectorproduction.

[0312] A gene therapy DNA construct can then be introduced into cells(either ex vivo or in vivo) using viral or non-viral vectors. One meansfor introducing the gene therapy DNA construct is by means of viralvectors as described herein. Certain vectors, such as retroviralvectors, will deliver the DNA construct to the chromosomal DNA of thecells, and the gene can integrate into the chromosomal DNA. Othervectors will function as episomes, and the gene therapy DNA constructwill remain in the cytoplasm.

[0313] In yet other embodiments, regulatory elements can be included forthe controlled expression of the TSLPR gene in the target cell. Suchelements are turned on in response to an appropriate effector. In thisway, a therapeutic polypeptide can be expressed when desired. Oneconventional control means involves the use of small molecule dimerizersor rapalogs to dimerize chimeric proteins which contain a smallmolecule-binding domain and a domain capable of initiating a biologicalprocess, such as a DNA-binding protein or transcriptional activationprotein (see PCT Pub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899).The dimerization of the proteins can be used to initiate transcriptionof the transgene.

[0314] An alternative regulation technology uses a method of storingproteins expressed from the gene of interest inside the cell as anaggregate or cluster. The gene of interest is expressed as a fusionprotein that includes a conditional aggregation domain that results inthe retention of the aggregated protein in the endoplasmic reticulum.The stored proteins are stable and inactive inside the cell. Theproteins can be released, however, by administering a drug (e.g., smallmolecule ligand) that removes the conditional aggregation domain andthereby specifically breaks apart the aggregates or clusters so that theproteins may be secreted from the cell. See Aridor et al., 2000, Science287:816-17 and Rivera et al., 2000, Science 287:826-30.

[0315] Other suitable control means or gene switches include, but arenot limited to, the systems described herein. Mifepristone (RU486) isused as a progesterone antagonist. The binding of a modifiedprogesterone receptor ligand-binding domain to the progesteroneantagonist activates transcription by forming a dimer of twotranscription factors that then pass into the nucleus to bind DNA. Theligand-binding domain is modified to eliminate the ability of thereceptor to bind to the natural ligand. The modified steroid hormonereceptor system is further described in U.S. Pat. No. 5,364,791 and PCTPub. Nos. WO 96/40911 and WO 97/10337.

[0316] Yet another control system uses ecdysone (a fruit fly steroidhormone) which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain, DNA-bindingdomain, and ligand-binding domain to initiate transcription. Theecdysone system is further described in U.S. Pat. No. 5,514,578 and PCTPub. Nos. WO 97/38117, WO 96/37609, and WO 93/03162.

[0317] Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758, 5,650,298, and 5,654,168.

[0318] Additional expression control systems and nucleic acid constructsare described in U.S. Pat. Nos. 5,741,679 and 5,834,186, to InnovirLaboratories Inc.

[0319] In vivo gene therapy may be accomplished by introducing the geneencoding TSLPR polypeptide into cells via local injection of a TSLPRnucleic acid molecule or by other appropriate viral or non-viraldelivery vectors. Hefti 1994, Neurobiology 25:1418-35. For example, anucleic acid molecule encoding a TSLPR polypeptide may be contained inan adeno-associated virus (AAV) vector for delivery to the targetedcells (see, e.g., Johnson, PCT Pub. No. WO 95/34670; PCT App. No.PCT/US95/07178). The recombinant AAV genome typically contains AAVinverted terminal repeats flanking a DNA sequence encoding a TSLPRpolypeptide operably linked to functional promoter and polyadenylationsequences.

[0320] Alternative suitable viral vectors include, but are not limitedto, retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Patent Nos. 5,631,236 (involvingadenoviral vectors), 5,672,510 (involving retroviral vectors), 5,635,399(involving retroviral vectors expressing cytokines).

[0321] Nonviral delivery methods include, but are not limited to,liposomic-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include induciblepromoters, tissue-specific enhancer-promoters, DNA sequences designedfor site-specific integration, DNA sequences capable of providing aselective advantage over the parent cell, labels to identify transformedcells, negative selection systems and expression control systems (safetymeasures), cell-specific binding agents (for cell targeting),cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as methods of vector manufacture.Such additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. Nos. 4,970,154 (involvingelectroporation techniques), 5,679,559 (describing alipoprotein-containing system for gene delivery), 5,676,954 (involvingliposome carriers), 5,593,875 (describing methods for calcium phosphatetransfection), and 4,945,050 (describing a process wherein biologicallyactive particles are propelled at cells at a speed whereby the particlespenetrate the surface of the cells and become incorporated into theinterior of the cells), and PCT Pub. No. WO 96/40958 (involving nuclearligands).

[0322] It is also contemplated that TSLPR gene therapy or cell therapycan further include the delivery of one or more additionalpolypeptide(s) in the same or a different cell(s). Such cells may beseparately introduced into the patient, or the cells may be contained ina single implantable device, such as the encapsulating membranedescribed above, or the cells may be separately modified by means ofviral vectors.

[0323] A means to increase endogenous TSLPR polypeptide expression in acell via gene therapy is to insert one or more enhancer elements intothe TSLPR polypeptide promoter, where the enhancer elements can serve toincrease transcriptional activity of the TSLPR gene. The enhancerelements used will be selected based on the tissue in which one desiresto activate the gene-enhancer elements known to confer promoteractivation in that tissue will be selected. For example, if a geneencoding a TSLPR polypeptide is to be “turned on” in T-cells, the lckpromoter enhancer element may be used. Here, the functional portion ofthe transcriptional element b be added may be inserted into a fragmentof DNA containing the TSLPR polypeptide promoter (and optionally,inserted into a vector and/or 5′ and/or 3′ flanking sequences) usingstandard cloning techniques. This construct, known as a “homologousrecombination construct,” can then be introduced into the desired cellseither ex vivo or in vivo.

[0324] Gene therapy also can be used to decrease TSLPR polypeptideexpression by modifying the nucleotide sequence of the endogenouspromoter. Such modification is typically accomplished via homologousrecombination methods. For example, a DNA molecule containing all or aportion of the promoter of the TSLPR gene selected for inactivation canbe engineered to remove and/or replace pieces of the promoter thatregulate transcription. For example, the TATA box and/or the bindingsite of a transcriptional activator of the promoter may be deleted usingstandard molecular biology techniques; such deletion can inhibitpromoter activity thereby repressing the transcription of thecorresponding TSLPR gene. The deletion of the TATA box or thetranscription activator binding site in the promoter may be accomplishedby generating a DNA construct comprising all or the relevant portion ofthe TSLPR polypeptide promoter (from the same or a related species asthe TSLPR gene to be regulated) in which one or more of the TATA boxand/or transcriptional activator binding site nucleotides are mutatedvia substitution, deletion and/or insertion of one or more nucleotides.As a result, the TATA box and/or activator binding site has decreasedactivity or is rendered completely inactive. This construct, which alsowill typically contain at least about 500 bases of DNA that correspondto the native (endogenous) 5′ and 3′ DNA sequences adjacent to thepromoter segment that has been modified, may be introduced into theappropriate cells (either ex vivo or in vivo) either directly or via aviral vector as described herein. Typically, the integration of theconstruct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

[0325] Therapeutic Uses

[0326] TSLPR nucleic acid molecules, polypeptides, and agonists andantagonists thereof can be used to treat, diagnose, ameliorate, orprevent a number of diseases, disorders, or conditions, includingTSLP-related diseases, disorders, or conditions. TSLP-related diseases,disorders, or conditions may be related to B-cell development, T-celldevelopment, T-cell receptor gene rearrangement, or regulation of theStat5 transcription factor. Diseases caused by or mediated byundesirable levels of TSLP are encompassed within the scope of theinvention. Undesirable levels include excessive levels of TSLP andsub-normal levels of TSLP.

[0327] TSLPR polypeptide agonists and antagonists include thosemolecules that regulate TSLPR polypeptide activity and either increaseor decrease at least one activity of the mature form of the TSLPRpolypeptide. Agonists or antagonists may be co-factors, such as aprotein, peptide, carbohydrate, lipid, or small molecular weightmolecule, which interact with TSLPR polypeptide and thereby regulate itsactivity. Potential polypeptide agonists or antagonists includeantibodies that react with either soluble or membrane-bound forms ofTSLPR polypeptides that comprise part or all of the extracellulardomains of the said proteins. Molecules that regulate TSLPR polypeptideexpression typically include nucleic acids encoding TSLPR polypeptidethat can act as anti-sense regulators of expression.

[0328] TSLPR nucleic acid molecules, polypeptides, and agonists andantagonists thereof may be used (simultaneously or sequentially) incombination with one or more cytokines, growth factors, antibiotics,anti-inflammatories, and/or chemotherapeutic agents as is appropriatefor the condition being treated.

[0329] Other diseases or disorders caused by or mediated by undesirablelevels of TSLPR polypeptides are encompassed within the scope of theinvention. Undesirable levels include excessive levels of TSLPRpolypeptides and sub-normal levels of TSLPR polypeptides.

[0330] Uses of TSLPR Nucleic Acids and Polypeptides

[0331] Nucleic acid molecules of the invention (including those that donot themselves encode biologically active polypeptides) may be used tomap the locations of the TSLPR gene and related genes on chromosomes.Mapping may be done by techniques known in the art, such as PCRamplification and in situ hybridization.

[0332] TSLPR nucleic acid molecules (including those that do notthemselves encode biologically active polypeptides) may be useful ashybridization probes in diagnostic assays to test, either qualitativelyor quantitatively, for the presence of a TSLPR nucleic acid molecule inmammalian tissue or bodily fluid samples.

[0333] Other methods may also be employed where it is desirable toinhibit the activity of one or more TSLPR polypeptides. Such inhibitionmay be effected by nucleic acid molecules that are complementary to andhybridize to expression control sequences (triple helix formation) or toTSLPR mRNA. For example, antisense DNA or RNA molecules, which have asequence that is complementary to at least a portion of a TSLPR gene canbe introduced into the cell. Anti-sense probes may be designed byavailable techniques using the sequence of the TSLPR gene disclosedherein. Typically, each such antisense molecule will be complementary tothe start site (5′ end) of each selected TSLPR gene. When the antisensemolecule then hybridizes to the corresponding TSLPR mRNA, translation ofthis mRNA is prevented or reduced. Anti-sense inhibitors provideinformation relating to the decrease or absence of a TSLPR polypeptidein a cell or organism.

[0334] Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more TSLPR polypeptides. In thissituation, the DNA encoding a mutant polypeptide of each selected TSLPRpolypeptide can be prepared and introduced into the cells of a patientusing either viral or non-viral methods as described herein. Each suchmutant is typically designed to compete with endogenous polypeptide inits biological role.

[0335] In addition, a TSLPR polypeptide, whether biologically active ornot, may be used as an immunogen, that is, the polypeptide contains atleast one epitope to which antibodies may be raised. Selective bindingagents that bind to a TSLPR polypeptide (as described herein) may beused for in vivo and in vitro diagnostic purposes, including, but notlimited to, use in labeled form to detect the presence of TSLPRpolypeptide in a body fluid or cell sample. The antibodies may also beused to prevent, treat, or diagnose a number of diseases and disorders,including those recited herein. The antibodies may bind to a TSLPRpolypeptide so as to diminish or block at least one activitycharacteristic of a TSLPR polypeptide, or may bind to a polypeptide toincrease at least one activity characteristic of a TSLPR polypeptide(including by increasing the pharmacokinetics of the TSLPR polypeptide).

[0336] The murine and human TSLPR nucleic acids of the present inventionare also useful tools for isolating the corresponding chromosomal TSLPRpolypeptide genes. For example, mouse chromosomal DNA containing TSLPRsequences can be used to construct knockout mice, thereby permitting anexamination of the in vivo role for TSLPR polypeptide. The human TSLPRgenomic DNA can be used to identify heritable tissue-degeneratingdiseases.

[0337] The following examples are intended for illustration purposesonly, and should not be construed as limiting the scope of the inventionin any way.

EXAMPLE 1 Cloning of the Murine and Human TSLPR Polypeptide Genes

[0338] Generally, materials and methods as described in Sambrook et al.,supra were used to clone and analyze the genes encoding murine and humanTSLPR polypeptides.

[0339] Sequences encoding the murine TSLPR polypeptide were identifiedin a BLAST search of an EST database using sequences corresponding tothe cytoplasmic domain of the erythropoietin receptor. Severaloverlapping murine ESTs, which encode a novel type I cytokine receptormolecule, were obtained in the BLAST search. The cytoplasmic domain ofthe cytokine receptor encoded by these sequences was found to sharesignificant similarity to that of the common cytokine receptor γ chain(γ_(c)), the erythropoietin receptor, and the IL-9 receptor α chain.

[0340] The common cytokine receptor γ chain is an essential subunit ofthe receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (Noguchi et al.,1993, Science 262:1877-80; Kondo et al., 1994, Science 263:1453-54;Kondo et al., 1993, Science 262:1874-77; Russell et al, 1994, Science266:1042-45; Takeshita et al., 1992, Science 257:379-82; Russell et al.,1993, Science 262:1880-83; Giri et al., 1994, EMBO J. 13:2822-30; Kimuraet al., 1995, Int. Immunol. 7:115-20). The mutation of γ_(c) in humanscan result in X-linked severe combined immunodeficiency (Noguchi et al.,1993, Cell 73:147-57; Leonard et al., 1995, Immunol. Rev. 148:97-114).

[0341] Since none of the ESTs sequences identified in the BLAST searchcontained the entire open reading frame for TSLPR polypeptide, a mouseembryo library was screened to obtain a full-length cDNA. The positivecolony containing the longest insert was used to prepare plasmid DNA bystandard methods. The cDNA insert from this colony was 2 kb in length.DNA sequence analysis confirmed that the clone contained the entirereading frame for TSLPR polypeptide.

[0342] Sequence analysis of the full-length cDNA for murine TSLPRpolypeptide indicated that the gene comprises a 1110 bp open readingframe encoding a protein of 370 amino acids and possessing a potentialsignal peptide of 17 amino acids in length at its amino-terminus (FIGS.1A-1B; predicted signal peptide indicated by underline). The openreading frame was found to encode a type I transmembrane protein havingtwo potential N-linked glycosylation sites and a cytoplasmic domain of104 amino acids containing a single tyrosine residue.

[0343] In contrast, murine γ_(c) comprises 369 amino acids a has acytoplasmic domain of 86 amino acids containing two tyrosine residues(Kumaki et al., 1993, Biochem. Biophys. Res. Commun. 193:356-63; Cao etal., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:8464-68; Kobayash et al.,1993, Gene 130:303-04). FIG. 2 illustrates an amino acid sequencealignment of murine TSLPR polypeptide (upper sequence) and murine γ_(c)(lower sequence). Murine TSLPR polypeptide was found to share 26%sequence identity and 47% sequence similarity with γ_(c) at the aminoacid level. The sequence of murine TSLPR polypeptide is somewhatatypical for type I cytokine receptors in that only one pair ofcysteines is conserved and the W-S-X-W-S (SEQ ID NO: 15) motif isreplaced by a W-T-A-V-T (SEQ ID NO: 16) motif. The predicted molecularweight of murine TSLPR polypeptide is 37 kD.

[0344] Sequences encoding the human TSLPR polypeptide were identified ina BLAST search of a proprietary database of cDNA sequences (Amgen,Thousand Oaks, Calif.) using the murine TSLPR nucleic acid sequence as aquery sequence. Two clones containing human cDNA sequences and sharingthe greatest homology with the murine TSLPR nucleic acid sequence wereidentified in this search: 9604927 (SEQ ID NO: 10) and 9508990 (SEQ IDNO: 11). Sequence analysis of the full-length cDNA for human TSLPRpolypeptide (as contained in Clone 9604927) indicated that the humanTSLPR gene comprises an open reading frame of 1113 bp encoding a proteinof 371 amino acids and possessing a potential signal peptide of 22 aminoacids in length at its amino-terminus (FIGS. 3A-3B; predicted signalpeptide indicated by underline).

[0345] Clone 9508990 contains an open reading frame of 1137 bp encodinga protein of 379 amino acids (FIGS. 4A-4B). This clone essentiallycomprises the full-length human TSLPR polypeptide sequence and anadditional 8 amino acids at the carboxyl-terminus corresponding to theFLAG epitope. FIG. 5 illustrates an amino acid sequence alignment ofmurine TSLPR polypeptide (upper sequence) and human TSLPR polypeptide(lower sequence). The availability of murine and human TSLPR nucleicacid and amino acid sequences will further aid in the elucidation ofsignal transduction pathways utilized by TSLP.

EXAMPLE 2 TSLPR Polypeptide Expression

[0346] A cDNA construct encoding the entire open reading frame formurine TSLPR was transcribed and translated in vitro in the presence of³⁵S-methionine and the product resolved by SDS-PAGE. FIG. 6A illustratesan autoradiogram of the gel in which a single species of approximately40 kD was obtained.

[0347]FIG. 6B illustrates the immunoprecipitation of murine TSLPRpolypeptide in the growth factor-dependent pre-B-cell line NAG8/7 usinga rabbit polyclonal antiserum raised against the extracellular domain ofmurine TSLPR polypeptide. The rabbit polyclonal antiserum was generatedagainst murine TSLPR polypeptide-glutathione S-transferase fusionprotein which was cloned into the pGEX4T2 expression vector (Pharmacia)and expressed in bacteria. Prior to metabolic labeling, NAG8/7 cellswere grown in RPMI supplemented with 10% fetal bovine serum,antibiotics, and TSLP.

[0348] NAG8/7 cells were metabolically labeled with ³⁵S-methionine andcysteine, lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, andprotease inhibitors, and the lysates incubated overnight with eitherrabbit polyclonal antiserum (lane 2) or pre-immune serum (lane 1). Theimmune complexes were captured with Protein G sepharosel, washed inlysis buffer, and then resolved by SDS-PAGE. The polyclonal antiserumspecifically immunoprecipitated a broad band of approximately 50 kD in apre-B-cell line NAG8/7 (FIG. 6B). The larger size of theimmunoprecipitated product as compared with the product generated by invitro translation is consistent with the addition of N-linkedcarbohydrate moieties in the extracellular domain. Flow cytometricanalysis of transfected 293 cells and several hematopoietic cell lines(i.e., 32D, BaF3, and WEHI-3) confirmed that murine TSLPR was expressedat the cell surface.

EXAMPLE 3 TSLPR mRNA Expression

[0349] The tissue distribution of murine TSLPR was examined by northernblot analysis. A mouse multiple tissue northern blot (Clontech, PaloAlto, Calif.) was screened with a ³²P-labeled TSLPR cDNA probe usingstandard techniques. Murine TSLPR mRNA transcripts were detected innearly all of the tissues examined, with highest levels of expressionbeing detected in the lung, liver, and testis (FIG. 6C). Lower levels ofexpression were detected in the heart, brain, spleen, and skeletalmuscle. Two transcripts of approximately 2 kb and 2.2 kb were detectedin some tissues, whereas only a single transcript of approximately 2 kbwas detected in other tissues. The broad tissue distribution of murineTSLPR mRNA differs from the relatively restricted lympho-hematopoieticpattern of expression observed forγ_(c).

[0350] The expression of TSLPR mRNA can be localized by in situhybridization as follows. A panel of normal embryonic and adult mousetissues is fixed in 4% paraformaldehyde, embedded in paraffin, andsectioned at 5 μm. Sectioned tissues are permeabilized in 0.2 M HCl,digested with Proteinase K, and acetylated with triethanolamine andacetic anhydride. Sections are prehybridized for 1 hour at 60° C. inhybridization solution (300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA,1X Denhardt's solution, 0.2% SDS, 10 mM DTT, 0.25 mg/ml tRNA, 25 μg/mlpolyA, 25 μg/ml polyC and 50% formamide) and then hybridized overnightat 60° C. in the same solution containing 10% dextran and 2×10⁴ cpm/μlof a ³³P-labeled antisense riboprobe complementary to the human TSLPRgene. The riboprobe is obtained by in vitro transcription of a clonecontaining human TSLPR EDNA sequences using standard techniques.

[0351] Following hybridization, sections are rinsed in hybridizationsolution, treated with RNaseA to digest unhybridized probe, and thenwashed in 0.1X SSC at 55° C. for minutes. Sections are then immersed inNTB-2 emulsion (Kodak, Rochester, N.Y.), exposed for 3 weeks at 4° C.,developed, and counterstained with hematoxylin and eosin. Tissuemorphology and hybridization signal are simultaneously analyzed bydarkfield and standard illumination for brain (one sagittal and twocoronal sections), gastrointestinal tract (esophagus, stomach, duodenum,jejunum, ileum, proximal colon, and distal colon), pituitary, liver,lung, heart, spleen, thymus, lymph nodes, kidney, adrenal, bladder,pancreas, salivary gland, male and female reproductive organs (ovary,oviduct, and uterus in the female; and testis, epididymus, prostate,seminal vesicle, and vas deferens in the male), BAT and WAT(subcutaneous, peri- renal), bone (femur), skin, breast, and skeletalmuscle.

EXAMPLE 4 Biological Activity of Murine TSLPR Polypeptide

[0352] The similarity between murine TSLPR polypeptide and theerythropoietin receptor suggested that murine TSLPR, like theerythropoietin receptor, could be activated by homodimerization. Thiswas examined in a proliferation assay using a chimeric construct derivedfrom the extracellular and transmembrane domains of the c-Kit receptorand the cytoplasmic domain of murine TSLPR polypeptide. To generate thisconstruct, the extracellular and transmembrane domains of c-Kit and thecytoplasmic domain of TSLPR were amplified by PCR and ligated into theretroviral vector pMX-IRES-GFP using standard techniques.

[0353] IL-2-dependent CTLL2 cells were stably transfected withexpression constructs encoding c-Kit/TSLPR and c-Kit/β, c-Kitβ andc-Kit/γ, or c-Kit/γ alone. The constructs for c-Kit/β and c-Kit/γ wereas described by Nelson et al., 1994, Nature 369:333-36. Followingtransfection, CTLL2 cells were deprived of IL-2, transferred into48-well dishes at 10,000 cells/well, and grown in the absence orpresence of Stem Cell Factor (SCF), the ligand for c-Kit. Cells werecounted after 7 days of growth in culture.

[0354]FIG. 7 illustrates that when IL-2 was replaced by SCF, CTLL2 cellsstably expressing chimeric c-Kit/TSLPR polypeptide were unable to grow,suggesting that simple homodimerization of the cytoplasmic domain ofmurine TSLPR polypeptide is insufficient to induce a proliferativesignal. Similar results have been obtained in proliferation experimentsusing a chimeric c-Kit/γ_(c) polypeptide (Nelson et al., supra).Furthermore, when CTLL2 cells were co-transfected with c-Kit/TSLPR andc-Kit/β, the cells were still unable to proliferate. However, CTLL2cells co-transfected with c-Kit/β and c-Kit/γ were able to proliferatefollowing incubation with SCF. This suggested that the cytoplasmicdomain of the IL-2Rβ chain could not cooperate with the cytoplasmicdomain of murine TSLPR polypeptide to initiate proliferation, and thatmurine TSLPR polypeptide might oligomerize with some other receptor toparticipate in signal transduction.

[0355] The similarity between murine TSLPR polypeptide and γ_(c)suggested that murine TSLPR may have the capacity to bind to some of themembers of the IL-2 cytokine subfamily. This was examined in an affinitylabeling assay using 125I-labeled IL-2, IL-4, IL-7, and IL-15. Prior tothe addition of an ¹²⁵I-labeled cytokine, 293 cells were reconstitutedwith the cytokine specific subunits IL-2Rβ, IL-4Rα, or IL-7Rα in thepresence of either γ_(c) or murine TSLPR polypeptide. None of theligands examined exhibited binding when murine TSLPR was co-expressedwith a cytokine specific subunit, even though the ligands efficientlybound when γ_(c) was co-expressed with a cytokine specific subunit. Thissuggested that murine TSLPR polypeptide either bound a novel cytokine orbound a known cytokine in conjunction with a novel or untested subunit.

[0356] Thymic stromal lymphopoietin (TSLP) is a cytokine whosebiological activities overlap with those of IL-7. TSLP activity wasoriginally identified in the conditioned medium of a thymic stromal cellline that supported the development of murine IgM⁺ B-cells from fetalliver hematopoietic progenitor cells (Friend et al., 1994 Exp. Hematol.22:321-28). Moreover, TSLP can promote B-cell lymphopoiesis in long-termbone marrow cultures and can co-stimulate both thymocytes and matureT-cells (Friend et al., supra; Levin et al., 1999, J. Immunol.162:67783).

[0357] Although IL-7 also possesses these activities (Suda et al., 1989,Blood 74:1936-41; Lee et al., 1989, J. Immunol. 142:3875-83; Sudo etal., 1989, J. Exp. Med. 170:333-38), TSLP is unique in that it promotesB lymphopoiesis to the IgM⁺ immature B-cell stage, while IL-7 primarilyfacilitates production of IgM⁻ pre-B-cells (Levin et al., supra;Candeias et al., 1997, Immunity 6:501-08). One possible explanation forthe overlapping biological activities of IL-7 and TSLP is that TSLPsignals via a receptor containing the IL-7Rα chain (Levin et al.,supra). However, antibody inhibition experiments have indicated thatTSLP does not require γ_(c) to exert its effects (Levin et al., supra).These results suggested that TSLP would bind murine TSLPR polypeptide inthe presence of IL-7Rα.

[0358] The binding of TSLP to TSLPR polypeptide in the presence ofIL-7Rα was examined in affinity labeling assays. Affinity labelingassays were performed by adding 1-5 nM of ¹²⁵I-labeled TSLP to 5×10⁶ 293cells transfected with expression constructs for murine IL-7Rα, murineTSLPR polypeptide, murine IL-7Rα and murine TSLPR polypeptide, or humanIL-7Rα and murine TSLPR polypeptide. Iodinated TSLP was prepared byadding IODO-GEN (Pierce, Rockford, Ill.) and 2 mCi¹²⁵I to 1 μg of TSLP.A specific activity of approximately 200-300 μCi/μg was obtained by thismethod. Prior to affinity labeling, 293 cells were transientlytransfected using the calcium phosphate method (Eppendorf-5 Prime,Boulder, Colo.). Following a 2 hour incubation with ¹²⁵I-TSLP, cellswere cross-linked with 0.1 mg/ml disuccinimidyl suberate (Pierce), lysedin lysis buffer, and the lysates resolved by SDS-PAGE.

[0359] As shown in FIG. 8A, ¹²⁵I-TSLP bound to the heterodimer of murineIL-7Rα and murine TSLPR polypeptide (lane 4). The upper band correspondsto cross-linked murine IL-7Rα and the lower band corresponds tocross-linked murine TSLPR polypeptide. In addition, ¹²⁵I-TSLP also boundthe heterodimer of human IL-7Rα and murine TSLPR polypeptide (lane 5).No TSLP binding was observed with murine IL-7Rα alone (lane 2).

[0360] Affinity labeling assays were also performed using a FLAG-taggedversion of murine TSLPR polypeptide. Murine TSLPR-FLAG polypeptide wasderived by PCR amplifying a fragment containing the coding region ofTSLPR polypeptide using a 3′ primer containing sequence corresponding tothe FLAG epitope. This PCR product was then subcloned into pCR3.1(Invitrogen) and the resulting clone analyzed by sequencing. Affinitylabeling assays were performed as described herein, with the exceptionthat cell lysates were immunoprecipitated with an anti-FLAG monoclonalM2 antibody. As shown in FIG. 8B, following TSLPR immunoprecipitation, across-linked TSLPR band was observed (lane 1), indicating that TSLPexhibits weak binding to TSLPR alone.

[0361] To examine whether murine IL-7 could compete for TSLP binding incells expressing TSLPR polypeptide and IL-7Rα, competition assays wereperformed. Cellular lysates were analyzed as described herein, with theexception that increasing amounts of unlabeled murine IL-7 were addedwith ¹²⁵I-TSLP. As shown in FIG. 8C, an excess of murine IL-7 inhibitedthe binding of TSLP to the IL-7Rα/TSLPR polypeptide heterodimer. Theaffinity labeling assays illustrated the cooperativity of IL-7Rα andmurine TSLPR polypeptide for binding TSLP. These assays also establishedthat IL-7 can compete for the binding of TSLP, which has implicationsfor potential competition between these two cytokines in vivo.

[0362] The binding of TSLP to 293 cells transfected with murine IL-7Rαand murine TSLPR polypeptide, or murine IL-7Rα alone, was analyzed in adisplacement binding assay. Following two washes, 1×10⁶ transfected 293cells were incubated in a constant amount of ¹²⁵I-labeled TSLP(approximately 20,000 cpm) and varying amounts of unlabeled TSLP.Following a 3 hour incubation, treated cells were separated from themedium by centrifugation in olive oil and N-butylphthalate. Cell-boundradioactivity was measured using a gamma counter.

[0363] As shown in FIG. 9A, non-specific binding of ¹²⁵I-TSLP wasobserved with cells transfected with murine IL-7Rα alone (or vectoralone), while specific binding of ¹²⁵I-TSLP was observed with cellstransfected with both IL-7Rα and TSLPR polypeptide, with excessunlabeled TSLP competing for binding of ¹²⁵I-TSLP. Cells transfectedwith TSLPR polypeptide alone exhibited very low binding. Analysis ofbinding data by Scatchard transformation was performed using the LIGANDcomputer program (Munson and Rodbard, 1980, Anal. Biochem. 107:220-39).The K_(d) for the binding of TSLP to cells expressing TSLPR polypeptideand IL-7Rα was determined to be approximately 13 nM (FIG. 9B). In sevenindependent experiments, the K_(d) was found to range from 1.2 to 40 nM.Due to the very low binding activity of TSLP for cells expressing TSLPRpolypeptide alone, it was not possible to determine the K_(d) for thesecells. Displacement binding assays were also performed using NAG8/7cells, which constitutively express TSLP receptors and proliferate inresponse to TSLP (Friend et al., supra; Levin et al., supra). In thesedisplacement binding assays, 5×10⁶ NAG8/7 cells were incubated in aconstant amount of ¹²⁵I-labeled TSLP (approximately 180,000 cpm) andvarying amounts of unlabeled TSLP. The remainder of the assay wasperformed as described herein. As shown in FIG. 9C, the Scatchardtransformation of binding data obtained using NAG8/7 cells suggested thecells expressed a single class of receptors having a K_(d) ofapproximately 2.2 nM-results that are similar to those obtained usingthe transfected 293 cells.

[0364] Displacement binding assays were also performed to compare thedisplacement of ¹²⁵I-labeled TSLP by IL-7 or unlabeled TSLP in 293 cellstransfected with TSLPR polypeptide and IL-7Rα. FIG. 9D illustrates thatmurine IL-7 competes for binding to TSLPR polypeptide.

[0365] It has been previously shown that treatment of NAG8/7 cells witheither IL-7 or TSLP activates STAT5 (Friend et al., supra; Levin et al.,supra). The possible role of TSLPR polypeptide in STAT5 activation wasanalyzed in CAT assays using HepG2 cells. Expression constructs forIL-7Rα and TSLPR, or IL-7Rα and γ_(c), were introduced into HepG2 cellswith the pHRRE-CAT vector by calcium phosphate transfection. ThepHRRE-CAT vector contains eight tandem copies of the 27 bpcytokine-inducible hematopoietin receptor response element and STAT5b(Ziegler et al., 1995, Eur. J. Immunol. 25:399-404). Transfected cellswere allowed to recover overnight, after which the cells weretrypsinized and plated in 6-well culture dishes. The cells were allowedto adhere to the plates during a 24 hour incubation, and the cells werethen incubated in serum free medium containing 100 ng/ml of either IL-7or TSLP, for an additional 24 hours.

[0366] The CAT activity and fold stimulation after normalizing fortransfection efficiencies is shown in FIG. 10. No increase in CATactivity was seen after TSLP stimulation in the presence of IL-7Rα alone(lane 2) or with IL-7Rα and γ_(c) (lane 7). However, if TSLPRpolypeptide was co-transfected, a dramatic increase in CAT activity wasobserved following TSLP stimulation (lane 5). This demonstrates that thepresence of TSLPR polypeptide is required for TSLP signaling. Whileco-transfection of γ_(c) and IL-7Rα had no effect on TSLP-dependentreporter activity, this combination effectively mediated IL-7-dependentreporter activation (lane 9).

[0367] A number of cytokine receptor chains are shared by more than onecytokine. The best known examples are gp130, which is shared by IL-6,IL-11, ciliary neurotropic factor, leukemia inhibitory factor,oncostatin M, and cardiotrophin-1 (Hirano et al., 1997, Cytokine GrowthFactor Rev. 8:241-52; Taga and Kishimoto, 1997, Annu. Rev. Immunol.15:797-819), β_(c), which is shared by IL-3, IL-5, and GM-CSF (Miyajimaet al., 1997, Leukemia 11:418-22; Guthridge et al., 1998, Stem Cells16:301-13; Burdach et al., 1998, Curr. Opin. Hematol. 5:177-80), andγ_(c), which is shared by IL-2, IL-4, IL-7, IL-9, and IL-15 (Noguchi etal., 1993, Science 262:1877-80; Kondo et al., 1994, supra; Kondo et al.,1993, supra; Russell et al., 1994, supra; Takeshita et al., supra;Russell et al., 1993, supra; Giri et al., supra; Kimura et al., supra).The list of cytokine receptor chains that serve as components of morethan one cytokine receptor includes IL-2Rβ, which is a component of boththe IL-2 and IL-receptors, and IL-4Rα, which is a component of both theIL-4 and IL-13 receptors. The cytokine receptor subunit IL-7Rα can nowbe added to this list as the data presented herein demonstrates thatthis subunit is a component of both the IL-7 and TSLP receptors.

[0368] The observation of defects in T-cell and B-cell development inI17^(l−) mice (von Freeden-Jeffrey et al., 1995, J. Exp. Med.181:1519-26) suggests that TSLP cannot fully compensate for the loss ofIL-7. An examination of the functional cooperation of IL-7Rα in TSLPsignaling may help to explain the differences in B-cell development inI17r^(−l−) and I17^(−l−) mice (Candeias et al., 1997, Immunity 6:501-08;von Freeden-Jeffrey et al., supra; Peschon et al., 1994, J. Exp. Med.180:1955-60; He et al., 1997, J. Immunol. 158:2592-99). The furthercharacterization of TSLPR polypeptide will aid this investigation.

EXAMPLE 5 Production of TSLPR Polypeptides

[0369] A. Expression of TSLPR Polypeptides in Bacteria

[0370] PCR is used to amplify template DNA sequences encoding a TSLPRpolypeptide using primers corresponding to the 5′ and 3′ ends of thesequence. The amplified DNA products may be modified to containrestriction enzyme sites to allow for insertion into expression vectors.PCR products are gel purified and inserted into expression vectors usingstandard recombinant DNA methodology. An exemplary vector, such aspAMG21 (ATCC no. 98113) containing the lux promoter and a gene encodingkanamycin resistance is digested with Bam HI and Nde I for directionalcloning of inserted DNA. The ligated mixture is transformed into an E.coli host strain by electroporation and transformants are selected forkanamycin resistance. Plasmid DNA from selected colonies is isolated andsubjected to DNA sequencing to confirm the presence of the insert.

[0371] Transformed host cells are incubated in 2xYT medium containing 30μg/mL kanamycin at 30° C. prior to induction. Gene expression is inducedby the addition of N-(3-oxohexanoyl)-dl-homoserine lactone to a finalconcentration of 30 ng/mL followed by incubation at either 30° C. or 37°C. for six hours. The expression of TSLPR polypeptide is evaluated bycentrifugation of the culture, resuspension and lysis of the bacterialpellets, and analysis of host cell proteins by SDS-polyacrylamide gelelectrophoresis.

[0372] Inclusion bodies containing TSLPR polypeptide are purified asfollows. Bacterial cells are pelleted by centrifugation and resuspendedin water. The cell suspension is lysed by sonication and pelleted bycentrifugation at 195,000 xg for 5 to 10 minutes. The supernatant isdiscarded, and the pellet is washed and transferred to a homogenizer.The pellet is homogenized in 5 mL of a Percoll solution (75% liquidPercoll and 0.15 M NaCl) until uniformly suspended and then diluted andcentrifuged at 21,600 xg for 30 minutes. Gradient fractions containingthe inclusion bodies are recovered and pooled. The isolated inclusionbodies are analyzed by SDS-PAGE.

[0373] A single band on an SDS polyacrylamide gel corresponding to E.coli-produced TSLPR polypeptide is excised from the gel, and theN-terminal amino acid sequence is determined essentially as described byMatsudaira et al., 1987, J. Biol. Chem. 262:10-35.

[0374] B. Expression of TSLPR Polypeptide in Mammalian Cells

[0375] PCR is used to amplify template DNA sequences encoding a TSLPRpolypeptide using primers corresponding to the 5′ and 3′ ends of thesequence. The amplified DNA products may be modified to containrestriction enzyme sites to allow for insertion into expression vectors.PCR products are gel purified and inserted into expression vectors usingstandard recombinant DNA methodology. An exemplary expression vector,pCEP4 (Invitrogen, Carlsbad, Calif.), that contains an Epstein-Barrvirus origin of replication, may be used for the expression of TSLPRpolypeptides in 293-EBNA-1 cells. Amplified and gel purified PCRproducts are ligated into pCEP4 vector and introduced into 293-EBNAcells by lipofection. The transfected cells are selected in 100 μg/mLhygromycin and the resulting drug-resistant cultures are grown toconfluence. The cells are then cultured in serum-free media for 72hours. The conditioned media is removed and TSLPR polypeptide expressionis analyzed by SDS-PAGE.

[0376] TSLPR polypeptide expression may be detected by silver staining.Alternatively, TSLPR polypeptide is produced as a fusion protein with anepitope tag, such as an IgG constant domain or a FLAG epitope, which maybe detected by Western blot analysis using antibodies to the peptidetag.

[0377] TSLPR polypeptides may be excised from an SDS-polyacrylamide gel,or TSLPR fusion proteins are purified by affinity chromatography to theepitope tag, and subjected to N-terminal amino acid sequence analysis asdescribed herein.

[0378] C. Expression and Purification of TSLPR Polypeptide in MammalianCells

[0379] TSLPR polypeptide expression constructs are introduced into 293EBNA or CHO cells using either a lipofection or calcium phosphateprotocol.

[0380] To conduct functional studies on the TSLPR polypeptides that areproduced, large quantities of conditioned media are generated from apool of hygromycin selected 293 EBNA clones. The cells are cultured in500 cm Nunc Triple Flasks to 80% confluence before switching to serumfree media a week prior to harvesting the media. Conditioned media isharvested and frozen at −20° C. until purification.

[0381] Conditioned media is purified by affinity chromatography asdescribed below. The media is thawed and then passed through a 0.2 μmfilter. A Protein G column is equilibrated with PBS at pH 7.0, and thenloaded with the filtered media. The column is washed with PBS until theabsorbance at A₂₈₀ reaches a baseline. TSLPR polypeptide is eluted fromthe column with 0.1 M Glycine-HCl at pH 2.7 and immediately neutralizedwith 1 M Tris-HCl at pH 8.5. Fractions containing TSLPR polypeptide arepooled, dialyzed in PBS, and stored at −70° C.

[0382] For Factor Xa cleavage of the human TSLPR polypeptide-Fc fusionpolypeptide, affinity chromatography-purified protein is dialyzed in 50mM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂ at pH 8.0. The restriction proteaseFactor Xa is added to the dialyzed protein at 1/100 (w/w) and the sampledigested overnight at room temperature.

EXAMPLE 6 Production of Anti-TSLPR Polypeptide Antibodies

[0383] Antibodies to TSLPR polypeptides may be obtained by immunizationwith purified protein or with TSLPR peptides produced by biological orchemical synthesis. Suitable procedures for generating antibodiesinclude those described in Hudson and Bay, Practical Immunology (2nded., Blackwell Scientific Publications).

[0384] In one procedure for the production of antibodies, animals(typically mice or rabbits) are injected with a TSLPR antigen (such as aTSLPR polypeptide), and those with sufficient serum titer levels asdetermined by ELISA are selected for hybridoma production. Spleens ofimmunized animals are collected and prepared as single cell suspensionsfrom which splenocytes are recovered. The splenocytes are fused to mousemyeloma cells (such as Sp2/0-Ag14 cells), are first incubated in DMEMwith 200 U/mL penicillin, 200 μg/mL streptomycin sulfate, and 4 mMglutamine, and are then incubated in HAT selection medium (hypoxanthine,aminopterin, and thymidine). After selection, the tissue culturesupernatants are taken from each fusion well and tested for anti-TSLPRantibody production by ELISA.

[0385] Alternative procedures for obtaining anti-TSLPR antibodies mayalso be employed, such as the immunization of transgenic mice harboringhuman Ig loci for production of human antibodies, and the screening ofsynthetic antibody libraries, such as those generated by mutagenesis ofan antibody variable domain.

EXAMPLE 7 Expression of TSLPR Polypeptide in Transgenic Mice

[0386] To assess the biological activity of TSLPR polypeptide, aconstruct encoding a TSLPR polypeptide/Fc fusion protein under thecontrol of a liver specific ApoE promoter is prepared. The delivery ofthis construct is expected to cause pathological changes that areinformative as to the function of TSLPR polypeptide. Similarly, aconstruct containing the full-length TSLPR polypeptide under the controlof the beta actin promoter is prepared. The delivery of this constructis expected to result in ubiquitous expression.

[0387] To generate these constructs, PCR is used to amplify template DNAsequences encoding a TSLPR polypeptide using primers that correspond tothe 5′ and 3′ ends of the desired sequence and which incorporaterestriction enzyme sites to permit insertion of the amplified productinto an expression vector. Following amplification, PCR products are gelpurified, digested with the appropriate restriction enzymes, and ligatedinto an expression vector using standard recombinant DNA techniques. Forexample, amplified TSLPR polypeptide sequences can be cloned into anexpression vector under the control of the human β-actin promoter asdescribed by Graham et al., 1997, Nature Genetics, 17:272-74 and Ray etal, 1991, Genes Dev. 5:2265-73.

[0388] Following ligation, reaction mixtures are used to transform an E.coli host strain by electroporation and transformants are selected fordrug resistance. Plasmid DNA from selected colonies is isolated andsubjected to DNA sequencing to confirm the presence of an appropriateinsert and absence of mutation. The TSLPR polypeptide expression vectoris purified through two rounds of CsCl density gradient centrifugation,cleaved with a suitable restriction enzyme, and the linearized fragmentcontaining the TSLPR polypeptide transgene is purified by gelelectrophoresis. The purified fragment is resuspended in 5 mM Tris, pH7.4, and 0.2 mM EDTA at a concentration of 2 mg/mL.

[0389] Single-cell embryos from BDF1×BDF1 bred mice are injected asdescribed (PCT Pub. No. WO 97/23614). Embryos are cultured overnight ina CO₂ incubator and 15-20 two-cell embryos are transferred to theoviducts of a pseudopregnant CD1 female mice. Offspring obtained fromthe implantation of microinjected embryos are screened by PCRamplification of the integrated transgene in genomic DNA samples asfollows. Ear pieces are digested in 20 mL ear buffer (20 mM Tris, pH8.0, 10 mM EDTA, 0.5% SDS, and 500 mg/mL proteinase K) at 55° C.overnight. The sample is then diluted with 200 mL of TE, and 2 mL of theear sample is used in a PCR reaction using appropriate primers.

[0390] At 8 weeks of age, transgenic founder animals and control animalsare sacrificed for necropsy and pathological analysis. Portions ofspleen are removed and total cellular RNA isolated from the spleensusing the Total RNA Extraction Kit (Qiagen) and transgene expressiondetermined by RT-PCR. RNA recovered from spleens is converted to cDNAusing the SuperScrip™ Preamplification System (Gibco-BRL) as follows. Asuitable primer, located in the expression vector sequence and 3′ to theTSLPR polypeptide transgene, is used to prime cDNA synthesis from thetransgene transcripts. Ten mg of total spleen RNA from transgenicfounders and controls is incubated with 1 mM of primer for 10 minutes at70° C. and placed on ice. The reaction is then supplemented with 10 mMTris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 10 mM of each dNTP, 0.1 mMDTT, and 200 U of SuperScript II reverse transcriptase. Followingincubation for 50 minutes at 42° C., the reaction is stopped by heatingfor 15 minutes at 72° C. and digested with 2U of RNase H for 20 minutesat 37° C. Samples are then amplified by PCR using primers specific forTSLPR polypeptide.

[0391] Determining the phenotypes of Tslp^(−l−) or Tslpr^(−l−) mice willalso assist in defining the exact role of TSLP.

EXAMPLE 8 Biological Activity of TSLPR Polypeptide in Transgenic Mice

[0392] Prior to euthanasia, transgenic animals are weighed, anesthetizedby isofluorane and blood drawn by cardiac puncture. The samples aresubjected to hematology and serum chemistry analysis. Radiography isperformed after terminal exsanguination. Upon gross dissection, majorvisceral organs are subject to weight analysis.

[0393] Following gross dissection, tissues (i.e., liver, spleen,pancreas, stomach, the entire gastrointestinal tract, kidney,reproductive organs, skin and mammary glands, bone, brain, heart, lung,thymus, trachea, esophagus, thyroid, adrenals, urinary bladder, lymphnodes and skeletal muscle) are removed and fixed in 10% bufferedZn-Formalin for histological examination. After fixation, the tissuesare processed into paraffin blocks, and 3 mm sections are obtained. Allsections are stained with hematoxylin and exosin, and are then subjectedto histological analysis.

[0394] The spleen, lymph node, and Peyer's patches of both thetransgenic and the control mice are subjected to immunohistologyanalysis with B cell and T cell specific antibodies as follows. Theformalin fixed paraffin embedded sections are deparaffinized andhydrated in deionized water. The sections are quenched with 3% hydrogenperoxide, blocked with Protein Block (Lipshaw, Pittsburgh, Pa.), andincubated in rat monoclonal anti-mouse B220 and CD3 (Harlan,Indianapolis, IN). Antibody binding is detected by biotinylated rabbitanti-rat immunoglobulins and peroxidase conjugated streptavidin(BioGenex, San Ramon, Calif.) with DAB as a chromagen (BioTek, SantaBarbara, Calif.). Sections are counterstained with hematoxylin.

[0395] After necropsy, MLN and sections of spleen and thymus fromtransgenic animals and control littermates are removed. Single cellsuspensions are prepared by gently grinding the tissues with the flatend of a syringe against the bottom of a 100 mm nylon cell strainer(Becton Dickinson, Franklin Lakes, N.J.). Cells are washed twice,counted, and approximately 1×10⁶ cells from each tissue are thenincubated for 10 minutes with 0.5 μg CD16/32(FcyIII/II) Fe block in a 20μL volume. Samples are then stained for 30 minutes at 2-8° C. in a 100μL volume of PBS (lacking Ca⁺ and Mg⁺), 0.1% bovine serum albumin, and0.01% sodium azide with 0.5 μg antibody of FITC or PE-conjugatedmonoclonal antibodies against CD90.2 (Thy-1.2), CD45R (B220), CD11b(Mac-1), Gr-1, CD4, or CD8 (PharMingen, San Diego, Calif.). Followingantibody binding, the cells are washed and then analyzed by flowcytometry on a FACScan (Becton Dickinson).

[0396] While the present invention has been described in terms of thepreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationsthat come within the scope of the invention as claimed.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 16 <210> SEQ ID NO 1<211> LENGTH: 1409 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (162)..(1274) <221>NAME/KEY: sig_peptide <222> LOCATION: (162)..(213) <221> NAME/KEY:misc_feature <222> LOCATION: (891)..(953) <223> OTHER INFORMATION:Predicted transmembrane domain coding sequence <400> SEQUENCE: 1ccccttcctc gccgacccct gaccccgccc cgccccgccc acccaggggc ccagacctga 60gcggcggcca ggtcgcgggt gacgtcacag ggccgttgcc ccatccgtcc cgtggcctgg 120acggacagag ctgaggcagg ggaataaccg cgagtgctga g atg gca tgg gca ctc 176Met Ala Trp Ala Leu 1 5 gcg gtc atc ctc ctg cct cgg ctc ctt gcg gcg gcagcg gcg gcg gcg 224 Ala Val Ile Leu Leu Pro Arg Leu Leu Ala Ala Ala AlaAla Ala Ala 10 15 20 gcg gtg acg tca cgg ggt gat gtc aca gtc gtc tgc catgac ctg gag 272 Ala Val Thr Ser Arg Gly Asp Val Thr Val Val Cys His AspLeu Glu 25 30 35 acg gtg gag gtc acg tgg ggc tcg ggc ccc gac cac cac agcgcc aac 320 Thr Val Glu Val Thr Trp Gly Ser Gly Pro Asp His His Ser AlaAsn 40 45 50 ttg agc ctg gag ttc cgt tat ggt act ggc gcc ctg caa ccc tgcccg 368 Leu Ser Leu Glu Phe Arg Tyr Gly Thr Gly Ala Leu Gln Pro Cys Pro55 60 65 cga tat ttc ctg tcc ggc gct ggt gtc act tcc ggg tgc atc ctc ccc416 Arg Tyr Phe Leu Ser Gly Ala Gly Val Thr Ser Gly Cys Ile Leu Pro 7075 80 85 gcg gcg agg gcg ggg ctg ctg gag ctg gca ctg cgc gac gga ggc ggg464 Ala Ala Arg Ala Gly Leu Leu Glu Leu Ala Leu Arg Asp Gly Gly Gly 9095 100 gcc atg gtg ttt aag gct agg cag cgc gcg tcc gcc tgg ctg aag ccc512 Ala Met Val Phe Lys Ala Arg Gln Arg Ala Ser Ala Trp Leu Lys Pro 105110 115 cgc cca cct tgg aat gtg acg ctg ctc tgg aca cca gac ggg gac gtg560 Arg Pro Pro Trp Asn Val Thr Leu Leu Trp Thr Pro Asp Gly Asp Val 120125 130 act gtc tcc tgg cct gcc cac tcc tac ctg ggc ctg gac tac gag gtg608 Thr Val Ser Trp Pro Ala His Ser Tyr Leu Gly Leu Asp Tyr Glu Val 135140 145 cag cac cgg gag agc aat gac gat gag gac gcc tgg cag acg acc tca656 Gln His Arg Glu Ser Asn Asp Asp Glu Asp Ala Trp Gln Thr Thr Ser 150155 160 165 ggg ccc tgc tgt gac ttg aca gtg ggc ggg ctc gac ccc gcg cgctgc 704 Gly Pro Cys Cys Asp Leu Thr Val Gly Gly Leu Asp Pro Ala Arg Cys170 175 180 tat gac ttc cgg gtt cgg gcg tcg ccc cgg gcc gcg cac tat ggcctg 752 Tyr Asp Phe Arg Val Arg Ala Ser Pro Arg Ala Ala His Tyr Gly Leu185 190 195 gag gcg cag cct agc gag tgg aca gcg gtg aca agg ctt tcc ggggca 800 Glu Ala Gln Pro Ser Glu Trp Thr Ala Val Thr Arg Leu Ser Gly Ala200 205 210 gca tcc gcg ggt gac ccc tgc gcc gcc cac ctt ccc ccc cta gcctcc 848 Ala Ser Ala Gly Asp Pro Cys Ala Ala His Leu Pro Pro Leu Ala Ser215 220 225 tgt acc gca agc ccc gcc cca tcc ccg gcc ctg gcc ccg ccc ctcctg 896 Cys Thr Ala Ser Pro Ala Pro Ser Pro Ala Leu Ala Pro Pro Leu Leu230 235 240 245 ccc ctg ggc tgc ggc cta gca gcg ctg ctg aca ctg tcc ctgctc ctg 944 Pro Leu Gly Cys Gly Leu Ala Ala Leu Leu Thr Leu Ser Leu LeuLeu 250 255 260 gcc gcc ctg agg ctt cgc agg gtg aaa gat gcg ctg ctg ccctgc gtc 992 Ala Ala Leu Arg Leu Arg Arg Val Lys Asp Ala Leu Leu Pro CysVal 265 270 275 cct gac ccc agc ggc tcc ttc cct gga ctc ttt gag aag catcac ggg 1040 Pro Asp Pro Ser Gly Ser Phe Pro Gly Leu Phe Glu Lys His HisGly 280 285 290 aac ttc cag gcc tgg att gcg gac gcc cag gcc aca gcc ccgcca gcc 1088 Asn Phe Gln Ala Trp Ile Ala Asp Ala Gln Ala Thr Ala Pro ProAla 295 300 305 agg acc gag gag gaa gat gac ctc atc cac ccc aag gct aagagg gtg 1136 Arg Thr Glu Glu Glu Asp Asp Leu Ile His Pro Lys Ala Lys ArgVal 310 315 320 325 gag ccc gag gat ggc acc tcc ctc tgc acc gtg cca aggcca ccc agc 1184 Glu Pro Glu Asp Gly Thr Ser Leu Cys Thr Val Pro Arg ProPro Ser 330 335 340 ttc gag cca agg ggg ccg gga ggc ggg gcc atg gtg tcagtg ggc ggg 1232 Phe Glu Pro Arg Gly Pro Gly Gly Gly Ala Met Val Ser ValGly Gly 345 350 355 gcc acg ttc atg gtg ggc gac agc ggc tac atg acc ctgtga 1274 Ala Thr Phe Met Val Gly Asp Ser Gly Tyr Met Thr Leu 360 365 370ccttgaagtc actgccagtc tatacttcag gctgaggtca cttcctgtct ttaaataatt 1334caaactcaca aatcctgtgc ctgtctgtat gcaaatgtgg tcacgaatat tcaaataaaa 1394tgcaaatgct atgct 1409 <210> SEQ ID NO 2 <211> LENGTH: 370 <212> TYPE:PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 2 Met Ala Trp Ala LeuAla Val Ile Leu Leu Pro Arg Leu Leu Ala Ala 1 5 10 15 Ala Ala Ala AlaAla Ala Val Thr Ser Arg Gly Asp Val Thr Val Val 20 25 30 Cys His Asp LeuGlu Thr Val Glu Val Thr Trp Gly Ser Gly Pro Asp 35 40 45 His His Ser AlaAsn Leu Ser Leu Glu Phe Arg Tyr Gly Thr Gly Ala 50 55 60 Leu Gln Pro CysPro Arg Tyr Phe Leu Ser Gly Ala Gly Val Thr Ser 65 70 75 80 Gly Cys IleLeu Pro Ala Ala Arg Ala Gly Leu Leu Glu Leu Ala Leu 85 90 95 Arg Asp GlyGly Gly Ala Met Val Phe Lys Ala Arg Gln Arg Ala Ser 100 105 110 Ala TrpLeu Lys Pro Arg Pro Pro Trp Asn Val Thr Leu Leu Trp Thr 115 120 125 ProAsp Gly Asp Val Thr Val Ser Trp Pro Ala His Ser Tyr Leu Gly 130 135 140Leu Asp Tyr Glu Val Gln His Arg Glu Ser Asn Asp Asp Glu Asp Ala 145 150155 160 Trp Gln Thr Thr Ser Gly Pro Cys Cys Asp Leu Thr Val Gly Gly Leu165 170 175 Asp Pro Ala Arg Cys Tyr Asp Phe Arg Val Arg Ala Ser Pro ArgAla 180 185 190 Ala His Tyr Gly Leu Glu Ala Gln Pro Ser Glu Trp Thr AlaVal Thr 195 200 205 Arg Leu Ser Gly Ala Ala Ser Ala Gly Asp Pro Cys AlaAla His Leu 210 215 220 Pro Pro Leu Ala Ser Cys Thr Ala Ser Pro Ala ProSer Pro Ala Leu 225 230 235 240 Ala Pro Pro Leu Leu Pro Leu Gly Cys GlyLeu Ala Ala Leu Leu Thr 245 250 255 Leu Ser Leu Leu Leu Ala Ala Leu ArgLeu Arg Arg Val Lys Asp Ala 260 265 270 Leu Leu Pro Cys Val Pro Asp ProSer Gly Ser Phe Pro Gly Leu Phe 275 280 285 Glu Lys His His Gly Asn PheGln Ala Trp Ile Ala Asp Ala Gln Ala 290 295 300 Thr Ala Pro Pro Ala ArgThr Glu Glu Glu Asp Asp Leu Ile His Pro 305 310 315 320 Lys Ala Lys ArgVal Glu Pro Glu Asp Gly Thr Ser Leu Cys Thr Val 325 330 335 Pro Arg ProPro Ser Phe Glu Pro Arg Gly Pro Gly Gly Gly Ala Met 340 345 350 Val SerVal Gly Gly Ala Thr Phe Met Val Gly Asp Ser Gly Tyr Met 355 360 365 ThrLeu 370 <210> SEQ ID NO 3 <211> LENGTH: 353 <212> TYPE: PRT <213>ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: TRANSMEM <222>LOCATION: (227)..(247) <400> SEQUENCE: 3 Ala Ala Ala Ala Ala Val Thr SerArg Gly Asp Val Thr Val Val Cys 1 5 10 15 His Asp Leu Glu Thr Val GluVal Thr Trp Gly Ser Gly Pro Asp His 20 25 30 His Ser Ala Asn Leu Ser LeuGlu Phe Arg Tyr Gly Thr Gly Ala Leu 35 40 45 Gln Pro Cys Pro Arg Tyr PheLeu Ser Gly Ala Gly Val Thr Ser Gly 50 55 60 Cys Ile Leu Pro Ala Ala ArgAla Gly Leu Leu Glu Leu Ala Leu Arg 65 70 75 80 Asp Gly Gly Gly Ala MetVal Phe Lys Ala Arg Gln Arg Ala Ser Ala 85 90 95 Trp Leu Lys Pro Arg ProPro Trp Asn Val Thr Leu Leu Trp Thr Pro 100 105 110 Asp Gly Asp Val ThrVal Ser Trp Pro Ala His Ser Tyr Leu Gly Leu 115 120 125 Asp Tyr Glu ValGln His Arg Glu Ser Asn Asp Asp Glu Asp Ala Trp 130 135 140 Gln Thr ThrSer Gly Pro Cys Cys Asp Leu Thr Val Gly Gly Leu Asp 145 150 155 160 ProAla Arg Cys Tyr Asp Phe Arg Val Arg Ala Ser Pro Arg Ala Ala 165 170 175His Tyr Gly Leu Glu Ala Gln Pro Ser Glu Trp Thr Ala Val Thr Arg 180 185190 Leu Ser Gly Ala Ala Ser Ala Gly Asp Pro Cys Ala Ala His Leu Pro 195200 205 Pro Leu Ala Ser Cys Thr Ala Ser Pro Ala Pro Ser Pro Ala Leu Ala210 215 220 Pro Pro Leu Leu Pro Leu Gly Cys Gly Leu Ala Ala Leu Leu ThrLeu 225 230 235 240 Ser Leu Leu Leu Ala Ala Leu Arg Leu Arg Arg Val LysAsp Ala Leu 245 250 255 Leu Pro Cys Val Pro Asp Pro Ser Gly Ser Phe ProGly Leu Phe Glu 260 265 270 Lys His His Gly Asn Phe Gln Ala Trp Ile AlaAsp Ala Gln Ala Thr 275 280 285 Ala Pro Pro Ala Arg Thr Glu Glu Glu AspAsp Leu Ile His Pro Lys 290 295 300 Ala Lys Arg Val Glu Pro Glu Asp GlyThr Ser Leu Cys Thr Val Pro 305 310 315 320 Arg Pro Pro Ser Phe Glu ProArg Gly Pro Gly Gly Gly Ala Met Val 325 330 335 Ser Val Gly Gly Ala ThrPhe Met Val Gly Asp Ser Gly Tyr Met Thr 340 345 350 Leu <210> SEQ ID NO4 <211> LENGTH: 1116 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1116) <221> NAME/KEY:sig_peptide <222> LOCATION: (1)..(66) <221> NAME/KEY: misc_feature <222>LOCATION: (694)..(756) <223> OTHER INFORMATION: Predicted transmembranedomain coding sequence <400> SEQUENCE: 4 atg ggg cgg ctg gtt ctg ctg tgggga gct gcc gtc ttt ctg ctg gga 48 Met Gly Arg Leu Val Leu Leu Trp GlyAla Ala Val Phe Leu Leu Gly 1 5 10 15 ggc tgg atg gct ttg ggg caa ggagga gca gca gaa gga gta cag att 96 Gly Trp Met Ala Leu Gly Gln Gly GlyAla Ala Glu Gly Val Gln Ile 20 25 30 cag atc atc tac ttc aat tta gaa accgtg cag gtg aca tgg aat gcc 144 Gln Ile Ile Tyr Phe Asn Leu Glu Thr ValGln Val Thr Trp Asn Ala 35 40 45 agc aaa tac tcc agg acc aac ctg act ttccac tac aga ttc aac ggt 192 Ser Lys Tyr Ser Arg Thr Asn Leu Thr Phe HisTyr Arg Phe Asn Gly 50 55 60 gat gag gcc tat gac cag tgc acc aac tac cttctc cag gaa ggt cac 240 Asp Glu Ala Tyr Asp Gln Cys Thr Asn Tyr Leu LeuGln Glu Gly His 65 70 75 80 act tca ggg tgc ctc cta gac gca gag cag cgagac gac att ctc tat 288 Thr Ser Gly Cys Leu Leu Asp Ala Glu Gln Arg AspAsp Ile Leu Tyr 85 90 95 ttc tcc atc agg aat ggg acg cac ccc gtt ttc accgca agt cgc tgg 336 Phe Ser Ile Arg Asn Gly Thr His Pro Val Phe Thr AlaSer Arg Trp 100 105 110 atg gtt tat tac ctg aaa ccc agt tcc ccg aag cacgtg aga ttt tcg 384 Met Val Tyr Tyr Leu Lys Pro Ser Ser Pro Lys His ValArg Phe Ser 115 120 125 tgg cat cag gat gca gtg acg gtg acg tgt tct gacctg tcc tac ggg 432 Trp His Gln Asp Ala Val Thr Val Thr Cys Ser Asp LeuSer Tyr Gly 130 135 140 gat ctc ctc tat gag gtt cag tac cgg agc ccc ttcgac acc gag tgg 480 Asp Leu Leu Tyr Glu Val Gln Tyr Arg Ser Pro Phe AspThr Glu Trp 145 150 155 160 cag tcc aaa cag gaa aat acc tgc aac gtc accata gaa ggc ttg gat 528 Gln Ser Lys Gln Glu Asn Thr Cys Asn Val Thr IleGlu Gly Leu Asp 165 170 175 gcc gag aag tgt tac tct ttc tgg gtc agg gtgaag gct atg gag gat 576 Ala Glu Lys Cys Tyr Ser Phe Trp Val Arg Val LysAla Met Glu Asp 180 185 190 gta tat ggg cca gac aca tac cca agc gac tggtca gag gtg aca tgc 624 Val Tyr Gly Pro Asp Thr Tyr Pro Ser Asp Trp SerGlu Val Thr Cys 195 200 205 tgg cag aga ggc gag att cgg gat gcc tgt gcagag aca cca acg cct 672 Trp Gln Arg Gly Glu Ile Arg Asp Ala Cys Ala GluThr Pro Thr Pro 210 215 220 ccc aaa cca aag ctg tcc aaa ttt att tta atttcc agc ctg gcc atc 720 Pro Lys Pro Lys Leu Ser Lys Phe Ile Leu Ile SerSer Leu Ala Ile 225 230 235 240 ctt ctg atg gtg tct ctc ctc ctt ctg tcttta tgg aaa tta tgg aga 768 Leu Leu Met Val Ser Leu Leu Leu Leu Ser LeuTrp Lys Leu Trp Arg 245 250 255 gtg aag aag ttt ctc att ccc agc gtg ccagac ccg aaa tcc atc ttc 816 Val Lys Lys Phe Leu Ile Pro Ser Val Pro AspPro Lys Ser Ile Phe 260 265 270 ccc ggg ctc ttt gag ata cac caa ggg aacttc cag gag tgg atc aca 864 Pro Gly Leu Phe Glu Ile His Gln Gly Asn PheGln Glu Trp Ile Thr 275 280 285 gac acc cag aac gtg gcc cac ctc cac aagatg gca ggt gca gag caa 912 Asp Thr Gln Asn Val Ala His Leu His Lys MetAla Gly Ala Glu Gln 290 295 300 gaa agt ggc ccc gag gag ccc ctg gta gtccag ttg gcc aag act gaa 960 Glu Ser Gly Pro Glu Glu Pro Leu Val Val GlnLeu Ala Lys Thr Glu 305 310 315 320 gcc gag tct ccc agg atg ctg gac ccacag acc gag gag aaa gag gcc 1008 Ala Glu Ser Pro Arg Met Leu Asp Pro GlnThr Glu Glu Lys Glu Ala 325 330 335 tct ggg gga tcc ctc cag ctt ccc caccag ccc ctc caa ggc ggt gat 1056 Ser Gly Gly Ser Leu Gln Leu Pro His GlnPro Leu Gln Gly Gly Asp 340 345 350 gtg gtc aca atc ggg ggc ttc acc tttgtg atg aat gac cgc tcc tac 1104 Val Val Thr Ile Gly Gly Phe Thr Phe ValMet Asn Asp Arg Ser Tyr 355 360 365 gtg gcg ttg tga 1116 Val Ala Leu 370<210> SEQ ID NO 5 <211> LENGTH: 371 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 5 Met Gly Arg Leu Val Leu Leu Trp Gly Ala AlaVal Phe Leu Leu Gly 1 5 10 15 Gly Trp Met Ala Leu Gly Gln Gly Gly AlaAla Glu Gly Val Gln Ile 20 25 30 Gln Ile Ile Tyr Phe Asn Leu Glu Thr ValGln Val Thr Trp Asn Ala 35 40 45 Ser Lys Tyr Ser Arg Thr Asn Leu Thr PheHis Tyr Arg Phe Asn Gly 50 55 60 Asp Glu Ala Tyr Asp Gln Cys Thr Asn TyrLeu Leu Gln Glu Gly His 65 70 75 80 Thr Ser Gly Cys Leu Leu Asp Ala GluGln Arg Asp Asp Ile Leu Tyr 85 90 95 Phe Ser Ile Arg Asn Gly Thr His ProVal Phe Thr Ala Ser Arg Trp 100 105 110 Met Val Tyr Tyr Leu Lys Pro SerSer Pro Lys His Val Arg Phe Ser 115 120 125 Trp His Gln Asp Ala Val ThrVal Thr Cys Ser Asp Leu Ser Tyr Gly 130 135 140 Asp Leu Leu Tyr Glu ValGln Tyr Arg Ser Pro Phe Asp Thr Glu Trp 145 150 155 160 Gln Ser Lys GlnGlu Asn Thr Cys Asn Val Thr Ile Glu Gly Leu Asp 165 170 175 Ala Glu LysCys Tyr Ser Phe Trp Val Arg Val Lys Ala Met Glu Asp 180 185 190 Val TyrGly Pro Asp Thr Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys 195 200 205 TrpGln Arg Gly Glu Ile Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro 210 215 220Pro Lys Pro Lys Leu Ser Lys Phe Ile Leu Ile Ser Ser Leu Ala Ile 225 230235 240 Leu Leu Met Val Ser Leu Leu Leu Leu Ser Leu Trp Lys Leu Trp Arg245 250 255 Val Lys Lys Phe Leu Ile Pro Ser Val Pro Asp Pro Lys Ser IlePhe 260 265 270 Pro Gly Leu Phe Glu Ile His Gln Gly Asn Phe Gln Glu TrpIle Thr 275 280 285 Asp Thr Gln Asn Val Ala His Leu His Lys Met Ala GlyAla Glu Gln 290 295 300 Glu Ser Gly Pro Glu Glu Pro Leu Val Val Gln LeuAla Lys Thr Glu 305 310 315 320 Ala Glu Ser Pro Arg Met Leu Asp Pro GlnThr Glu Glu Lys Glu Ala 325 330 335 Ser Gly Gly Ser Leu Gln Leu Pro HisGln Pro Leu Gln Gly Gly Asp 340 345 350 Val Val Thr Ile Gly Gly Phe ThrPhe Val Met Asn Asp Arg Ser Tyr 355 360 365 Val Ala Leu 370 <210> SEQ IDNO 6 <211> LENGTH: 349 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: TRANSMEM <222> LOCATION: (210)..(230)<400> SEQUENCE: 6 Gln Gly Gly Ala Ala Glu Gly Val Gln Ile Gln Ile IleTyr Phe Asn 1 5 10 15 Leu Glu Thr Val Gln Val Thr Trp Asn Ala Ser LysTyr Ser Arg Thr 20 25 30 Asn Leu Thr Phe His Tyr Arg Phe Asn Gly Asp GluAla Tyr Asp Gln 35 40 45 Cys Thr Asn Tyr Leu Leu Gln Glu Gly His Thr SerGly Cys Leu Leu 50 55 60 Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr Phe SerIle Arg Asn Gly 65 70 75 80 Thr His Pro Val Phe Thr Ala Ser Arg Trp MetVal Tyr Tyr Leu Lys 85 90 95 Pro Ser Ser Pro Lys His Val Arg Phe Ser TrpHis Gln Asp Ala Val 100 105 110 Thr Val Thr Cys Ser Asp Leu Ser Tyr GlyAsp Leu Leu Tyr Glu Val 115 120 125 Gln Tyr Arg Ser Pro Phe Asp Thr GluTrp Gln Ser Lys Gln Glu Asn 130 135 140 Thr Cys Asn Val Thr Ile Glu GlyLeu Asp Ala Glu Lys Cys Tyr Ser 145 150 155 160 Phe Trp Val Arg Val LysAla Met Glu Asp Val Tyr Gly Pro Asp Thr 165 170 175 Tyr Pro Ser Asp TrpSer Glu Val Thr Cys Trp Gln Arg Gly Glu Ile 180 185 190 Arg Asp Ala CysAla Glu Thr Pro Thr Pro Pro Lys Pro Lys Leu Ser 195 200 205 Lys Phe IleLeu Ile Ser Ser Leu Ala Ile Leu Leu Met Val Ser Leu 210 215 220 Leu LeuLeu Ser Leu Trp Lys Leu Trp Arg Val Lys Lys Phe Leu Ile 225 230 235 240Pro Ser Val Pro Asp Pro Lys Ser Ile Phe Pro Gly Leu Phe Glu Ile 245 250255 His Gln Gly Asn Phe Gln Glu Trp Ile Thr Asp Thr Gln Asn Val Ala 260265 270 His Leu His Lys Met Ala Gly Ala Glu Gln Glu Ser Gly Pro Glu Glu275 280 285 Pro Leu Val Val Gln Leu Ala Lys Thr Glu Ala Glu Ser Pro ArgMet 290 295 300 Leu Asp Pro Gln Thr Glu Glu Lys Glu Ala Ser Gly Gly SerLeu Gln 305 310 315 320 Leu Pro His Gln Pro Leu Gln Gly Gly Asp Val ValThr Ile Gly Gly 325 330 335 Phe Thr Phe Val Met Asn Asp Arg Ser Tyr ValAla Leu 340 345 <210> SEQ ID NO 7 <211> LENGTH: 1140 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Human TSLPR-FLAG <221>NAME/KEY: CDS <222> LOCATION: (1)..(1140) <221> NAME/KEY: sig_peptide<222> LOCATION: (1)..(66) <221> NAME/KEY: misc_feature <222> LOCATION:(694)..(756) <223> OTHER INFORMATION: Predicted transmembrane domaincoding sequence <221> NAME/KEY: misc_feature <222> LOCATION:(1114)..(1140) <223> OTHER INFORMATION: FLAG coding sequence <400>SEQUENCE: 7 atg ggg cgg ctg gtt ctg ctg tgg gga gct gcc gtc ttt ctg ctggga 48 Met Gly Arg Leu Val Leu Leu Trp Gly Ala Ala Val Phe Leu Leu Gly 15 10 15 ggc tgg atg gct ttg ggg caa gga gga gca gca gaa gga gta cag att96 Gly Trp Met Ala Leu Gly Gln Gly Gly Ala Ala Glu Gly Val Gln Ile 20 2530 cag atc atc tac ttc aat tta gaa acc gtg cag gtg aca tgg aat gcc 144Gln Ile Ile Tyr Phe Asn Leu Glu Thr Val Gln Val Thr Trp Asn Ala 35 40 45agc aaa tac tcc agg acc aac ctg act ttc cac tac aga ttc aac ggt 192 SerLys Tyr Ser Arg Thr Asn Leu Thr Phe His Tyr Arg Phe Asn Gly 50 55 60 gatgag gcc tat gac cag tgc acc aac tac ctt ctc cag gaa ggt cac 240 Asp GluAla Tyr Asp Gln Cys Thr Asn Tyr Leu Leu Gln Glu Gly His 65 70 75 80 acttca ggg tgc ctc cta gac gca gag cag cga gac gac att ctc tat 288 Thr SerGly Cys Leu Leu Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr 85 90 95 ttc tccatc agg aat ggg acg cac ccc gtt ttc acc gca agt cgc tgg 336 Phe Ser IleArg Asn Gly Thr His Pro Val Phe Thr Ala Ser Arg Trp 100 105 110 atg gtttat tac ctg aaa ccc agt tcc ccg aag cac gtg aga ttt tcg 384 Met Val TyrTyr Leu Lys Pro Ser Ser Pro Lys His Val Arg Phe Ser 115 120 125 tgg catcag gat gca gtg acg gtg acg tgt tct gac ctg tcc tac ggg 432 Trp His GlnAsp Ala Val Thr Val Thr Cys Ser Asp Leu Ser Tyr Gly 130 135 140 gat ctcctc tat gag gtt cag tac cgg agc ccc ttc gac acc gag tgg 480 Asp Leu LeuTyr Glu Val Gln Tyr Arg Ser Pro Phe Asp Thr Glu Trp 145 150 155 160 cagtcc aaa cag gaa aat acc tgc aac gtc acc ata gaa ggc ttg gat 528 Gln SerLys Gln Glu Asn Thr Cys Asn Val Thr Ile Glu Gly Leu Asp 165 170 175 gccgag aag tgt tac tct ttc tgg gtc agg gtg aag gct atg gag gat 576 Ala GluLys Cys Tyr Ser Phe Trp Val Arg Val Lys Ala Met Glu Asp 180 185 190 gtatat ggg cca gac aca tac cca agc gac tgg tca gag gtg aca tgc 624 Val TyrGly Pro Asp Thr Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys 195 200 205 tggcag aga ggc gag att cgg gat gcc tgt gca gag aca cca acg cct 672 Trp GlnArg Gly Glu Ile Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro 210 215 220 cccaaa cca aag ctg tcc aaa ttt att tta att tcc agc ctg gcc atc 720 Pro LysPro Lys Leu Ser Lys Phe Ile Leu Ile Ser Ser Leu Ala Ile 225 230 235 240ctt ctg atg gtg tct ctc ctc ctt ctg tct tta tgg aaa tta tgg aga 768 LeuLeu Met Val Ser Leu Leu Leu Leu Ser Leu Trp Lys Leu Trp Arg 245 250 255gtg aag aag ttt ctc att ccc agc gtg cca gac ccg aaa tcc atc ttc 816 ValLys Lys Phe Leu Ile Pro Ser Val Pro Asp Pro Lys Ser Ile Phe 260 265 270ccc ggg ctc ttt gag ata cac caa ggg aac ttc cag gag tgg atc aca 864 ProGly Leu Phe Glu Ile His Gln Gly Asn Phe Gln Glu Trp Ile Thr 275 280 285gac acc cag aac gtg gcc cac ctc cac aag atg gca ggt gca gag caa 912 AspThr Gln Asn Val Ala His Leu His Lys Met Ala Gly Ala Glu Gln 290 295 300gaa agt ggc ccc gag gag ccc ctg gta gtc cag ttg gcc aag act gaa 960 GluSer Gly Pro Glu Glu Pro Leu Val Val Gln Leu Ala Lys Thr Glu 305 310 315320 gcc gag tct ccc agg atg ctg gac cca cag acc gag gag aaa gag gcc 1008Ala Glu Ser Pro Arg Met Leu Asp Pro Gln Thr Glu Glu Lys Glu Ala 325 330335 tct ggg gga tcc ctc cag ctt ccc cac cag ccc ctc caa ggc ggt gat 1056Ser Gly Gly Ser Leu Gln Leu Pro His Gln Pro Leu Gln Gly Gly Asp 340 345350 gtg gtc aca atc ggg ggc ttc acc ttt gtg atg aat gac cgc tcc tac 1104Val Val Thr Ile Gly Gly Phe Thr Phe Val Met Asn Asp Arg Ser Tyr 355 360365 gtg gcg ttg gac tac aag gac gac gat gac aag tag 1140 Val Ala Leu AspTyr Lys Asp Asp Asp Asp Lys 370 375 <210> SEQ ID NO 8 <211> LENGTH: 379<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Human TSLPR-FLAG<400> SEQUENCE: 8 Met Gly Arg Leu Val Leu Leu Trp Gly Ala Ala Val PheLeu Leu Gly 1 5 10 15 Gly Trp Met Ala Leu Gly Gln Gly Gly Ala Ala GluGly Val Gln Ile 20 25 30 Gln Ile Ile Tyr Phe Asn Leu Glu Thr Val Gln ValThr Trp Asn Ala 35 40 45 Ser Lys Tyr Ser Arg Thr Asn Leu Thr Phe His TyrArg Phe Asn Gly 50 55 60 Asp Glu Ala Tyr Asp Gln Cys Thr Asn Tyr Leu LeuGln Glu Gly His 65 70 75 80 Thr Ser Gly Cys Leu Leu Asp Ala Glu Gln ArgAsp Asp Ile Leu Tyr 85 90 95 Phe Ser Ile Arg Asn Gly Thr His Pro Val PheThr Ala Ser Arg Trp 100 105 110 Met Val Tyr Tyr Leu Lys Pro Ser Ser ProLys His Val Arg Phe Ser 115 120 125 Trp His Gln Asp Ala Val Thr Val ThrCys Ser Asp Leu Ser Tyr Gly 130 135 140 Asp Leu Leu Tyr Glu Val Gln TyrArg Ser Pro Phe Asp Thr Glu Trp 145 150 155 160 Gln Ser Lys Gln Glu AsnThr Cys Asn Val Thr Ile Glu Gly Leu Asp 165 170 175 Ala Glu Lys Cys TyrSer Phe Trp Val Arg Val Lys Ala Met Glu Asp 180 185 190 Val Tyr Gly ProAsp Thr Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys 195 200 205 Trp Gln ArgGly Glu Ile Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro 210 215 220 Pro LysPro Lys Leu Ser Lys Phe Ile Leu Ile Ser Ser Leu Ala Ile 225 230 235 240Leu Leu Met Val Ser Leu Leu Leu Leu Ser Leu Trp Lys Leu Trp Arg 245 250255 Val Lys Lys Phe Leu Ile Pro Ser Val Pro Asp Pro Lys Ser Ile Phe 260265 270 Pro Gly Leu Phe Glu Ile His Gln Gly Asn Phe Gln Glu Trp Ile Thr275 280 285 Asp Thr Gln Asn Val Ala His Leu His Lys Met Ala Gly Ala GluGln 290 295 300 Glu Ser Gly Pro Glu Glu Pro Leu Val Val Gln Leu Ala LysThr Glu 305 310 315 320 Ala Glu Ser Pro Arg Met Leu Asp Pro Gln Thr GluGlu Lys Glu Ala 325 330 335 Ser Gly Gly Ser Leu Gln Leu Pro His Gln ProLeu Gln Gly Gly Asp 340 345 350 Val Val Thr Ile Gly Gly Phe Thr Phe ValMet Asn Asp Arg Ser Tyr 355 360 365 Val Ala Leu Asp Tyr Lys Asp Asp AspAsp Lys 370 375 <210> SEQ ID NO 9 <211> LENGTH: 357 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Human TSLPR-FLAG <221>NAME/KEY: TRANSMEM <222> LOCATION: (210)..(230) <221> NAME/KEY: DOMAIN<222> LOCATION: (350)..(357) <223> OTHER INFORMATION: FLAG sequence<400> SEQUENCE: 9 Gln Gly Gly Ala Ala Glu Gly Val Gln Ile Gln Ile IleTyr Phe Asn 1 5 10 15 Leu Glu Thr Val Gln Val Thr Trp Asn Ala Ser LysTyr Ser Arg Thr 20 25 30 Asn Leu Thr Phe His Tyr Arg Phe Asn Gly Asp GluAla Tyr Asp Gln 35 40 45 Cys Thr Asn Tyr Leu Leu Gln Glu Gly His Thr SerGly Cys Leu Leu 50 55 60 Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr Phe SerIle Arg Asn Gly 65 70 75 80 Thr His Pro Val Phe Thr Ala Ser Arg Trp MetVal Tyr Tyr Leu Lys 85 90 95 Pro Ser Ser Pro Lys His Val Arg Phe Ser TrpHis Gln Asp Ala Val 100 105 110 Thr Val Thr Cys Ser Asp Leu Ser Tyr GlyAsp Leu Leu Tyr Glu Val 115 120 125 Gln Tyr Arg Ser Pro Phe Asp Thr GluTrp Gln Ser Lys Gln Glu Asn 130 135 140 Thr Cys Asn Val Thr Ile Glu GlyLeu Asp Ala Glu Lys Cys Tyr Ser 145 150 155 160 Phe Trp Val Arg Val LysAla Met Glu Asp Val Tyr Gly Pro Asp Thr 165 170 175 Tyr Pro Ser Asp TrpSer Glu Val Thr Cys Trp Gln Arg Gly Glu Ile 180 185 190 Arg Asp Ala CysAla Glu Thr Pro Thr Pro Pro Lys Pro Lys Leu Ser 195 200 205 Lys Phe IleLeu Ile Ser Ser Leu Ala Ile Leu Leu Met Val Ser Leu 210 215 220 Leu LeuLeu Ser Leu Trp Lys Leu Trp Arg Val Lys Lys Phe Leu Ile 225 230 235 240Pro Ser Val Pro Asp Pro Lys Ser Ile Phe Pro Gly Leu Phe Glu Ile 245 250255 His Gln Gly Asn Phe Gln Glu Trp Ile Thr Asp Thr Gln Asn Val Ala 260265 270 His Leu His Lys Met Ala Gly Ala Glu Gln Glu Ser Gly Pro Glu Glu275 280 285 Pro Leu Val Val Gln Leu Ala Lys Thr Glu Ala Glu Ser Pro ArgMet 290 295 300 Leu Asp Pro Gln Thr Glu Glu Lys Glu Ala Ser Gly Gly SerLeu Gln 305 310 315 320 Leu Pro His Gln Pro Leu Gln Gly Gly Asp Val ValThr Ile Gly Gly 325 330 335 Phe Thr Phe Val Met Asn Asp Arg Ser Tyr ValAla Leu Asp Tyr Lys 340 345 350 Asp Asp Asp Asp Lys 355 <210> SEQ ID NO10 <211> LENGTH: 1379 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Clone 9604927 containing human TSLPR sequence <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(68) <223> OTHERINFORMATION: Vector sequence <221> NAME/KEY: sig_peptide <222> LOCATION:(70)..(135) <221> NAME/KEY: misc_feature <222> LOCATION: (763)..(825)<223> OTHER INFORMATION: Predicted transmembrane domain coding sequence<221> NAME/KEY: misc_feature <222> LOCATION: (1186)..(1379) <223> OTHERINFORMATION: Vector sequence <400> SEQUENCE: 10 ggatccacta gtaacggccgccagtgtgct ggaattctgc agatatccat cacactggcg 60 gccgccacca tggggcggctggttctgctg tggggagctg ccgtctttct gctgggaggc 120 tggatggctt tggggcaaggaggagcagca gaaggagtac agattcagat catctacttc 180 aatttagaaa ccgtgcaggtgacatggaat gccagcaaat actccaggac caacctgact 240 ttccactaca gattcaacggtgatgaggcc tatgaccagt gcaccaacta ccttctccag 300 gaaggtcaca cttcagggtgcctcctagac gcagagcagc gagacgacat tctctatttc 360 tccatcagga atgggacgcaccccgttttc accgcaagtc gctggatggt ttattacctg 420 aaacccagtt ccccgaagcacgtgagattt tcgtggcatc aggatgcagt gacggtgacg 480 tgttctgacc tgtcctacggggatctcctc tatgaggttc agtaccggag ccccttcgac 540 accgagtggc agtccaaacaggaaaatacc tgcaacgtca ccatagaagg cttggatgcc 600 gagaagtgtt actctttctgggtcagggtg aaggctatgg aggatgtata tgggccagac 660 acatacccaa gcgactggtcagaggtgaca tgctggcaga gaggcgagat tcgggatgcc 720 tgtgcagaga caccaacgcctcccaaacca aagctgtcca aatttatttt aatttccagc 780 ctggccatcc ttctgatggtgtctctcctc cttctgtctt tatggaaatt atggagagtg 840 aagaagtttc tcattcccagcgtgccagac ccgaaatcca tcttccccgg gctctttgag 900 atacaccaag ggaacttccaggagtggatc acagacaccc agaacgtggc ccacctccac 960 aagatggcag gtgcagagcaagaaagtggc cccgaggagc ccctggtagt ccagttggcc 1020 aagactgaag ccgagtctcccaggatgctg gacccacaga ccgaggagaa agaggcctct 1080 gggggatccc tccagcttccccaccagccc ctccaaggcg gtgatgtggt cacaatcggg 1140 ggcttcacct ttgtgatgaatgaccgctcc tacgtggcgt tgtgatctaa agggccctat 1200 tctatactgt cacctaaatgctagagctcg ctgatcagcc tcgactgtgc cttctagttg 1260 ccagccatct gttgtttgcccctcccccgt gccttccttg accctggaat gtgccactcc 1320 cactgtcctt tcctaataaaatgaagaaat tgcatccgca ttgtctgagt aggtgtcta 1379 <210> SEQ ID NO 11 <211>LENGTH: 1415 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Clone 9508990 containing human TSLPR-FLAG sequence <221> NAME/KEY:misc_feature <222> LOCATION: (1)..(60) <223> OTHER INFORMATION: Vectorsequence <221> NAME/KEY: sig_peptide <222> LOCATION: (62)..(127) <221>NAME/KEY: misc_feature <222> LOCATION: (755)..(817) <223> OTHERINFORMATION: Predicted transmembrane domain coding sequence <221>NAME/KEY: misc_feature <222> LOCATION: (1175)..(1201) <223> OTHERINFORMATION: FLAG coding sequence <221> NAME/KEY: misc_feature <222>LOCATION: (1202)..(1415) <223> OTHER INFORMATION: Vector sequence <400>SEQUENCE: 11 ggatccacta gtaacggccg ccagtgtgct ggaattctgc agatatccatcacactggcc 60 catggggcgg ctggttctgc tgtggggagc tgccgtcttt ctgctgggaggctggatggc 120 tttggggcaa ggaggagcag cagaaggagt acagattcag atcatctacttcaatttaga 180 aaccgtgcag gtgacatgga atgccagcaa atactccagg accaacctgactttccacta 240 cagattcaac ggtgatgagg cctatgacca gtgcaccaac taccttctccaggaaggtca 300 cacttcaggg tgcctcctag acgcagagca gcgagacgac attctctatttctccatcag 360 gaatgggacg caccccgttt tcaccgcaag tcgctggatg gtttattacctgaaacccag 420 ttccccgaag cacgtgagat tttcgtggca tcaggatgca gtgacggtgacgtgttctga 480 cctgtcctac ggggatctcc tctatgaggt tcagtaccgg agccccttcgacaccgagtg 540 gcagtccaaa caggaaaata cctgcaacgt caccatagaa ggcttggatgccgagaagtg 600 ttactctttc tgggtcaggg tgaaggctat ggaggatgta tatgggccagacacataccc 660 aagcgactgg tcagaggtga catgctggca gagaggcgag attcgggatgcctgtgcaga 720 gacaccaacg cctcccaaac caaagctgtc caaatttatt ttaatttccagcctggccat 780 ccttctgatg gtgtctctcc tccttctgtc tttatggaaa ttatggagagtgaagaagtt 840 tctcattccc agcgtgccag acccgaaatc catcttcccc gggctctttgagatacacca 900 agggaacttc caggagtgga tcacagacac ccagaacgtg gcccacctccacaagatggc 960 aggtgcagag caagaaagtg gccccgagga gcccctggta gtccagttggccaagactga 1020 agccgagtct cccaggatgc tggacccaca gaccgaggag aaagaggcctctgggggatc 1080 cctccagctt ccccaccagc ccctccaagg cggtgatgtg gtcacaatcgggggcttcac 1140 ctttgtgatg aatgaccgct cctacgtggc gttggactac aaggacgacgatgacaagta 1200 gtctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctgatcagactcga 1260 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgccttccttgaccc 1320 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgcatcgcattgtc 1380 tgagtaggtg tcattctatt ctggggggtg gcgtt 1415 <210> SEQ IDNO 12 <211> LENGTH: 369 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 12 Met Leu Lys Leu Leu Leu Ser Pro Arg Ser Phe Leu ValLeu Gln Leu 1 5 10 15 Leu Leu Leu Arg Ala Gly Trp Ser Ser Lys Val LeuMet Ser Ser Ala 20 25 30 Asn Glu Asp Ile Lys Ala Asp Leu Ile Leu Thr SerThr Ala Pro Glu 35 40 45 His Leu Ser Ala Pro Thr Leu Pro Leu Pro Glu ValGln Cys Phe Val 50 55 60 Phe Asn Ile Glu Tyr Met Asn Cys Thr Trp Asn SerSer Ser Glu Pro 65 70 75 80 Gln Ala Thr Asn Leu Thr Leu His Tyr Arg TyrLys Val Ser Asp Asn 85 90 95 Asn Thr Phe Gln Glu Cys Ser His Tyr Leu PheSer Lys Glu Ile Thr 100 105 110 Ser Gly Cys Gln Ile Gln Lys Glu Asp IleGln Leu Tyr Gln Thr Phe 115 120 125 Val Val Gln Leu Gln Asp Pro Gln LysPro Gln Arg Arg Ala Val Gln 130 135 140 Lys Leu Asn Leu Gln Asn Leu ValIle Pro Arg Ala Pro Glu Asn Leu 145 150 155 160 Thr Leu Ser Asn Leu SerGlu Ser Gln Leu Glu Leu Arg Trp Lys Ser 165 170 175 Arg His Ile Lys GluArg Cys Leu Gln Tyr Leu Val Gln Tyr Arg Ser 180 185 190 Asn Arg Asp ArgSer Trp Thr Glu Leu Ile Val Asn His Glu Pro Arg 195 200 205 Phe Ser LeuPro Ser Val Asp Glu Leu Lys Arg Tyr Thr Phe Arg Val 210 215 220 Arg SerArg Tyr Asn Pro Ile Cys Gly Ser Ser Gln Gln Trp Ser Lys 225 230 235 240Trp Ser Gln Pro Val His Trp Gly Ser His Thr Val Glu Glu Asn Pro 245 250255 Ser Leu Phe Ala Leu Glu Ala Val Leu Ile Pro Val Gly Thr Met Gly 260265 270 Leu Ile Ile Thr Leu Ile Phe Val Tyr Cys Trp Leu Glu Arg Met Pro275 280 285 Pro Ile Pro Pro Ile Lys Asn Leu Glu Asp Leu Val Thr Glu TyrGln 290 295 300 Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu ThrGlu Ser 305 310 315 320 Leu Gln Pro Asp Tyr Ser Glu Arg Phe Cys His ValSer Glu Ile Pro 325 330 335 Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro GlyGly Ser Pro Cys Ser 340 345 350 Leu His Ser Pro Tyr Trp Pro Pro Pro CysTyr Ser Leu Lys Pro Glu 355 360 365 Ala <210> SEQ ID NO 13 <211> LENGTH:11 <212> TYPE: PRT <213> ORGANISM: Human immunodeficiency virus type 1<400> SEQUENCE: 13 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10<210> SEQ ID NO 14 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Internalizing domain derived from HIV tatprotein <400> SEQUENCE: 14 Gly Gly Gly Gly Tyr Gly Arg Lys Lys Arg ArgGln Arg Arg Arg 1 5 10 15 <210> SEQ ID NO 15 <211> LENGTH: 5 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Type I cytokinereceptor conserved motif <221> NAME/KEY: UNSURE <222> LOCATION: (3)<223> OTHER INFORMATION: “Xaa” can be any naturally occurring amino acid<400> SEQUENCE: 15 Trp Ser Xaa Trp Ser 1 5 <210> SEQ ID NO 16 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Motif replacing type I cytokine receptor conserved motif in murine TSLPRpolypeptide <400> SEQUENCE: 16 Trp Thr Ala Val Thr 1 5

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence selected from the group consisting of: (a) thenucleotide sequence as set forth in any of SEQ ID NO: 4, SEQ ID NO: 7,SEQ IDNO: 10, or SEQIDNO: 1; (b) a nucleotide sequence encoding thepolypeptide as set forth in either SEQ ID NO: 5 or SEQ ID NO: 8; (c) anucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of either (b) or (c); and (d) anucleotide sequence complementary to either (b) or (c).
 2. An isolatednucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence encoding a polypeptidewhich is at least about 70 percent identical to the polypeptide as setforth in either SEQ ID NO: 5 or SEQ ID NO: 8, wherein the encodedpolypeptide has an activity of the polypeptide set forth in either SEQID NO: 5 or SEQ ID NO: 8; (b) a nucleotide sequence encoding an allelicvariant or splice variant of the nucleotide sequence as set forth in anyof SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, or (a);(c) a region of the nucleotide sequence of any of SEQ ID NO: 4, SEQ IDNO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, (a), or (b) encoding apolypeptide fragment of at least about 25 amino acid residues, whereinthe polypeptide fragment has an activity of the encoded polypeptide asset forth in either SEQ ID NO: 5 or SEQ ID NO: 8, or is antigenic; (d) aregion of the nucleotide sequence of any of SEQ ID NO: 4, SEQ ID NO: 7,SEQ ID NO: 10, or SEQ ID NO: 11, or any of (a)-(c) comprising a fragmentof at least about 16 nucleotides; (e) a nucleotide sequence whichhybridizes under moderately or highly stringent conditions to thecomplement of any of (a)-(d); and (f) a nucleotide sequencecomplementary to any of (a)-(d).
 3. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence encoding a polypeptide as set forth in eitherSEQ ID NO: 5 or SEQ ID NO: 8 with at least one conservative amino acidsubstitution, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 5 or SEQ ID NO: 8; (b) anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 5 or SEQ ID NO: 8 with at least one amino acid insertion, whereinthe encoded polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 5 or SEQ ID NO: 8; (c) a nucleotide sequence encodinga polypeptide as set forth in either SEQ ID NO: 5 or SEQ ID NO: 8 withat least one amino acid deletion, wherein the encoded polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 5 or SEQ IDNO: 8; (d) a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 5 or SEQ ID NO: 8 which has a C- and/or N-terminaltruncation, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 5 or SEQ ID NO: 8; (e) anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 5 or SEQ ID NO: 8 with at least one modification selected from thegroup consisting of amino acid substitutions, amino acid insertions,amino acid deletions, C-terminal truncation, and N-terminal truncation,wherein the encoded polypeptide has an activity of the polypeptide setforth in either SEQ ID NO: 5 or SEQ ID NO: 8; (f) a nucleotide sequenceof any of (a)-(e) comprising a fragment of at least about 16nucleotides; (g) a nucleotide sequence which hybridizes under moderatelyor highly stringent conditions to the complement of any of (a)-(f); and(h) a nucleotide sequence complementary to any of (a)-(e).
 4. A vectorcomprising the nucleic acid molecule of any of claims 1, 2, or
 3. 5. Ahost cell comprising the vector of claim
 4. 6. The host cell of claim 5that is a eukaryotic cell.
 7. The host cell of claim 5 that is aprokaryotic cell.
 8. A process of producing a TSLPR polypeptidecomprising culturing the host cell of claim 5 under suitable conditionsto express the polypeptide, and optionally isolating the polypeptidefrom the culture.
 9. A polypeptide produced by the process of claim 8.10. The process of claim 8, wherein the nucleic acid molecule comprisespromoter DNA other than the promoter DNA for the native TSLPRpolypeptide operatively linked to the DNA encoding the TSLPRpolypeptide.
 11. The isolated nucleic acid molecule according to claim2, wherein the percent identity is determined using a computer programselected from the group consisting of GAP, BLASTN, FASTA, BLASTA,BLASTX, BestFit, and the Smith-Waterman algorithm.
 12. A process fordetermining whether a compound inhibits TSLPR polypeptide activity orTSLPR polypeptide production comprising exposing a cell according to anyof claims 5, 6, or 7 to the compound and measuring TSLPR polypeptideactivity or TSLPR polypeptide production in said cell.
 13. An isolatedpolypeptide comprising the amino acid sequence as set forth in eitherSEQ ID NO: 5 or SEQ ID NO:
 8. 14. An isolated polypeptide comprising theamino acid sequence selected from the group consisting of: (a) the aminoacid sequence as set forth in either SEQ ID NO: 6 or SEQ ID NO: 9,optionally further comprising an amino-terminal methionine; (b) an aminoacid sequence for an ortholog of either SEQ ID NO: 5 or SEQ ID NO: 8;(c) an amino acid sequence which is at least about 70 percent identicalto the amino acid sequence of either SEQ ID NO: 5 or SEQ ID NO: 8,wherein the polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 5 or SEQ ID NO: 8; (d) a fragment of the amino acidsequence set forth in either SEQ ID NO: 5 or SEQ ID NO: 8 comprising atleast about 25 amino acid residues, wherein the fragment has an activityof the polypeptide set forth in either SEQ ID NO: 5 or SEQ ID NO: 8, oris antigenic; and (e) an amino acid sequence for an allelic variant orsplice variant of the amino acid sequence as set forth in either SEQ IDNO: 5 or SEQ ID NO: 8, or any of (a)-(c).
 15. An isolated polypeptidecomprising the amino acid sequence selected from the group consistingof: (a) the amino acid sequence as set forth in either SEQ ID NO: 5 orSEQ ID NO: 8 with at least one conservative amino acid substitution,wherein the polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 5 or SEQ ID NO: 8; (b) the amino acid sequence as setforth in either SEQ ID NO: 5 or SEQ ID NO: 8 with at least one aminoacid insertion, wherein the polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 5 or SEQ ID NO: 8; (c) theamino acid sequence as set forth in either SEQ ID NO: 5 or SEQ ID NO: 8with at least one amino acid deletion, wherein the polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 5 or SEQ IDNO: 8; (d) the amino acid sequence as set forth in either SEQ ID NO: 5or SEQ ID NO: 8 which has a C- and/or N-terminal truncation, wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 5 or SEQ ID NO: 8; and (e) the amino acid sequence as set forthin either SEQ ID NO: 5 or SEQ ID NO: 8 with at least one modificationselected from the group consisting of amino acid substitutions, aminoacid insertions, amino acid deletions, C-terminal truncation, andN-terminal truncation, wherein the polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 5 or SEQ ID NO:
 8. 16. Anisolated polypeptide encoded by the nucleic acid molecule of any ofclaims 1, 2, or 3, wherein the polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 5 or SEQ ID NO:
 8. 17. Theisolated polypeptide according to claim 14, wherein the percent identityis determined using a computer program selected from the groupconsisting of GAP, BLASTP, FASTA, BLASTA, BLASTX, BestFit, and theSmith-Waterman algorithm.
 18. A selective binding agent or fragmentthereof that specifically binds the polypeptide of any of claims 13, 14,or
 15. 19. The selective binding agent or fragment thereof of claim 18that specifically binds the polypeptide comprising the amino acidsequence as set forth in either SEQ ID NO: 5 or SEQ ID NO: 8, or afragment thereof.
 20. The selective binding agent of claim 18 that is anantibody or fragment thereof.
 21. The selective binding agent of claim18 that is a humanized antibody.
 22. The selective binding agent ofclaim 18 that is a human antibody or fragment thereof.
 23. The selectivebinding agent of claim 18 that is a polyclonal antibody or fragmentthereof.
 24. The selective binding agent claim 18 that is a monoclonalantibody or fragment thereof.
 25. The selective binding agent of claim18 that is a chimeric antibody or fragment thereof.
 26. The selectivebinding agent of claim 18 that is a CDR-grafted antibody or fragmentthereof.
 27. The selective binding agent of claim 18 that is anantiidiotypic antibody or fragment thereof.
 28. The selective bindingagent of claim 18 that is a variable region fragment.
 29. The variableregion fragment of claim 28 that is a Fab or a Fab′ fragment.
 30. Aselective binding agent or fragment thereof comprising at least onecomplementarity determining region with specificity for a polypeptidehaving the amino acid sequence of either SEQ ID NO: 5 or SEQ ID NO: 8.31. The selective binding agent of claim 18 that is bound to adetectable label.
 32. The selective binding agent of claim 18 thatantagonizes TSLPR polypeptide biological activity.
 33. A method fortreating, preventing, or ameliorating a TSLPR polypeptide-relateddisease, condition, or disorder comprising administering to a patient aneffective amount of a selective binding agent according to claim
 18. 34.A selective binding agent produced by immunizing an animal with apolypeptide comprising an amino acid sequence of either SEQ ID NO: 5 orSEQ ID NO:
 8. 35. A hybridoma that produces a selective binding agentcapable of binding a polypeptide according to any of claims 1, 2, or 3.36. A method of detecting or quantitating the amount of TSLPRpolypeptide using the anti-TSLPR antibody or fragment of claim
 18. 37. Acomposition comprising the polypeptide of any of claims 13, 14, or 15,and a pharmaceutically acceptable formulation agent.
 38. The compositionof claim 37, wherein the pharmaceutically acceptable formulation agentis a carrier, adjuvant, solubilizer, stabilizer, or anti-oxidant. 39.The composition of claim 37 wherein the polypeptide comprises the aminoacid sequence as set forth in either SEQ ID NO: 6 or SEQ ID NO:
 9. 40. Apolypeptide comprising a derivative of the polypeptide of any of claims13, 14, or
 15. 41. The polypeptide of claim 40 that is covalentlymodified with a water-soluble polymer.
 42. The polypeptide of claim 41,wherein the water-soluble polymer is selected from the group consistingof polyethylene glycol, monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, and polyvinyl alcohol.
 43. A compositioncomprising a nucleic acid molecule of any of claims 1, 2, or 3 and apharmaceutically acceptable formulation agent.
 44. The composition ofclaim 43, wherein said nucleic acid molecule is contained in a viralvector.
 45. A viral vector comprising a nucleic acid molecule of any ofclaims 1, 2, or
 3. 46. A fusion polypeptide comprising the polypeptideof any of claims 13, 14, or 15 fused to a heterologous amino acidsequence.
 47. The fusion polypeptide of claim 46, wherein theheterologous amino acid sequence is an IgG constant domain or fragmentthereof.
 48. A method for treating, preventing, or ameliorating amedical condition comprising administering to a patient the polypeptideof any of claims 13, 14, or 15, or the polypeptide encoded by thenucleic acid of any of claims 1, 2, or
 3. 49. A method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising: (a) determining the presence or amount ofexpression of the polypeptide of any of claims 13, 14, or 15, or thepolypeptide encoded by the nucleic acid molecule of any of claims 1, 2,or 3 in a sample; and (b) diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence oramount of expression of the polypeptide.
 50. A device, comprising: (a) amembrane suitable for implantation; and (b) cells encapsulated withinsaid membrane, wherein said cells secrete a protein of any of claims 13,14, or 15; and said membrane is permeable to said protein andimpermeable to materials detrimental to said cells.
 51. A method ofidentifying a compound which binds to a TSLPR polypeptide comprising:(a) contacting the polypeptide of any of claims 13, 14, or 15 with acompound; and (b) determining the extent of binding of the TSLPRpolypeptide to the compound.
 52. The method of claim 51, furthercomprising determining the activity of the polypeptide when bound to thecompound.
 53. A method of modulating levels of a polypeptide in ananimal comprising administering to the animal the nucleic acid moleculeof any of claims 1, 2, or
 3. 54. A transgenic non-human mammalcomprising the nucleic acid molecule of any of claims 1, 2, or
 3. 55. Aprocess for determining whether a compound inhibits TSLPR polypeptideactivity or TSLPR polypeptide production comprising exposing atransgenic mammal according to claim 54 to the compound, and measuringTSLPR polypeptide activity or TSLPR polypeptide production in saidmammal.
 56. A nucleic acid molecule of any of claims 1, 2, or 3 attachedto a solid support.
 57. An array of nucleic acid molecules comprising atleast one nucleic acid molecule of any of claims 1, 2, or 3.